Compositions of Phosphorylated Tau Peptides and Uses Thereof

ABSTRACT

Liposomes containing tau peptides, preferably phosphorylated tau peptides, and conjugates containing tau peptides, preferably phosphorylated tau peptides, conjugated to an immunogenic carrier are described. Pharmaceutical compositions and uses of the liposomes and/or conjugates for treating or preventing a neurodegenerative disease or disorder, such as Alzheimer&#39;s Disease, are also described.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. application Ser. No. 16/169,215,filed on Oct. 24, 2018, which claims the benefit of U.S. ProvisionalApplication 62/577,157, filed on Oct. 25, 2017, the disclosure of eachof these prior applications is hereby incorporated by reference hereinin its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “SequenceListing2US4”, creation date of Jul. 28, 2021, andhaving a size of 22 KB. The sequence listing submitted via EFS-Web ispart of the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention is in the field of medicine. The invention inparticular relates to liposomes or conjugates of tau peptides and theuse thereof for preventing or treating tauopathy, such as Alzheimer'sdisease.

BACKGROUND

Alzheimer's disease (AD) is a progressive debilitating neurodegenerativedisease that affects an estimated 44 million people worldwide(Alzheimers.net). AD therapies that are currently available in theclinic aim to slow the progression of clinical symptoms, but do nottarget the pathogenic processes that underlie the disease.Unfortunately, these therapies are only minimally efficacious, and thereis therefore an urgent need to develop and test additional preventiveand therapeutic measures.

The hallmark pathologies for Alzheimer's disease are an accumulation ofextracellular plaques comprising aggregated amyloid beta protein andintracellular “tangles” or aggregations of hyperphosphorylated tauprotein. The molecular events that lead to accumulation of theseproteins are poorly characterized. For amyloid, it is hypothesized thataberrant cleavage of the amyloid precursor protein leads to anaccumulation of the aggregation-prone fragment comprising amino acids1-42. For tau, it is hypothesized that dysregulation of either kinases,phosphatases, or both, leads to aberrant phosphorylation of tau. Oncetau becomes hyperphosphorylated it loses the ability to effectively bindand stabilize microtubules, and instead accumulates in the cytoplasm ofthe affected neuron. The unbound and hyperphosphorylated tau appears toform first oligomers and then higher order aggregates, the presence ofwhich presumably negatively affects function of the neuron in which theyform, perhaps via interruption of normal axonal transport.

In developed nations, individuals diagnosed with Alzheimer's disease orother dementing tauopathies are commonly treated with cholinesteraseinhibitors (e.g. Aricept®) or memantines (e.g. Namenda™). These drugs,although reasonably well tolerated, have very modest efficacy. Forexample, Aricept® delays the worsening of symptoms for 6-12 months inapproximately 50% of treated individuals. The remainder of treatment isnon-pharmacologic, and focuses on making patients more capable ofmanaging day to day tasks as their cognitive ability declines.

Several published studies (Asuni A A et al, J Neurosci. 2007 Aug. 22;27(34):9115-29., Theunis C et al., PLoS One. 2013; 8(8): e72301.,Kontsekova E et al., Alzheimers Res Ther. 2014 Aug. 1; 6(4):44)demonstrate that active vaccines containing tau peptides can induceanti-tau immune responses in mice or rats; reduce the accumulation ofpathologic tau aggregates in the brain of rodents; and reduce the rateof progression of cognitive decline in animal models of Alzheimer'sdisease. An active vaccine against pathological tau proteins was shownto be immunogenic in human patients with Alzheimer's disease (Novak P etal., Lancet Neurology 2017, 16:123-134). WO2010/115843 describesantigenic phosphopeptide mimicking a major pathological phospho-epitopeof protein tau and related compositions for the therapeutic anddiagnostic use in the treatment of tauopathies including Alzheimer'sDisease. However, at present there are still no approved efficaciousvaccines on the market to prevent the onset of tau-mediated disease.Neither are there efficacious drugs on the market to intercept or slowthe course of disease once it begins. There is therefore a pressing needto identify new preventative measures (e.g. vaccines) that can preventthese diseases.

SUMMARY OF THE INVENTION

In one general aspect, the invention relates to a liposome, comprising:

a. a tau peptide, preferably the tau peptide is a tau phosphopeptide;and

b. a helper T-cell epitope,

wherein the tau peptide is presented on the surface of the liposome.

In one embodiment, the liposome further comprises at least one adjuvantcomprising a toll-like receptor ligand. Preferably, the liposome furthercomprises at least one of a toll-like receptor 4 ligand and a toll-likereceptor 9 ligand.

In a preferred embodiment, the invention relates to a liposome,comprising:

a. a tau peptide, preferably the tau peptide is a tau phosphopeptide;

b. a helper T-cell epitope; and

c. at least one of

-   -   i. a toll-like receptor 9 ligand, preferably a lipidated CpG        oligonucleotide; and    -   ii. a toll-like receptor 4 ligand, preferably a toll-like        receptor 4 agonist,

wherein the tau peptide is presented on the surface of the liposome.

In a further preferred embodiment, the invention relates to a liposome,comprising:

a. a tau phosphopeptide;

b. a helper T-cell epitope;

c. a lipidated CpG oligonucleotide; and

d. an adjuvant containing a toll-like receptor 4 ligand;

wherein the tau phosphopeptide is presented on the surface of theliposome.

In another general aspect, the invention relates to a conjugatecomprising a tau peptide, preferably a tau phosphopeptide, and animmunogenic carrier conjugated thereto, wherein the tau peptide isconjugated to the carrier via a linker. The linker can comprise one ormore of polyethylene glycol (PEG), succinimidyl3-(bromoacetamido)propionate (SBAP), andm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Examples of theimmunogenic carrier useful for the invention include, but are notlimited to, keyhole limpet hemocyanin (KLH), tetanus toxoid (TT),CRM197, and an outer membrane protein mixture from N. meningitidis(OMP), or a derivative thereof.

In one preferred embodiment, the invention relates to a conjugate havingthe structure of formula (I):

or the structure of formula (II):

wherein

x is an integer of 0 to 10, preferably 2 to 6, most preferably 3; and

n is an integer of 2 to 11, preferably 3 to 11.

Further aspects of the invention relate to a pharmaceutical compositioncomprising a liposome or a conjugate of the invention and apharmaceutically acceptable carrier, methods of preparing thepharmaceutical composition, and the use of the pharmaceuticalcomposition in inducing an immune response against tau, or treating orpreventing a neurodegenerative disease or disorder in a subject in needthereof.

In one embodiment, the invention relates to a method for inducing animmune response in a subject suffering from a neurodegenerativedisorder, or for treating or preventing a neurodegenerative disease ordisorder in a subject in need thereof. The method comprisesadministering to the subject a pharmaceutical composition comprising aliposome of the invention and a pharmaceutically acceptable carrier, ora pharmaceutical composition comprising a conjugate of the invention anda pharmaceutically acceptable carrier. Preferably, the method comprisesadministering to the subject a pharmaceutical composition of theinvention for priming immunization, and a pharmaceutical composition ofthe invention for boosting immunization.

Further aspects, features and advantages of the present invention willbe better appreciated upon a reading of the following detaileddescription of the invention and claims.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description ofpreferred embodiments of the present application, will be betterunderstood when read in conjunction with the appended drawings. Itshould be understood, however, that the application is not limited tothe precise embodiments shown in the drawings.

FIG. 1 illustrates novel vaccines according to embodiments of theinvention: a tau liposome according to an embodiment of the invention(top), and a tau conjugate according to an embodiment of the invention(bottom);

FIG. 2 illustrates that a vaccine comprising a liposome according to anembodiment of the invention (2nd generation liposome) which contains anencapsulated helper T-cell epitope (e.g., tetanus polypeptide (tet))activates helper T-cells;

FIG. 3 illustrates that a vaccine comprising a conjugate according to anembodiment of the invention which contains non-self or immunogeniccarrier protein activates helper T-cells;

FIG. 4 shows that tau vaccines according to embodiments of the inventioninduce sustained high titer anti-phosphorylated tau antibodies in Rhesusmacaque: the geometric mean of end-point titers per group, as measuredby enzyme-linked immunosorbent assay (ELISA), over time, is higher for avaccine comprising a liposome (Liposome Z) according to an embodiment ofthe invention or a vaccine comprising a conjugate (Conjugate A)according to an embodiment of the invention, as compared to a controlliposomal vaccine without the helper T-cell epitope;

FIG. 5 shows that serum from Rhesus macaques immunized with a liposome(Liposome Z) according to an embodiment of the invention binds topathological tau structures in human AD brain sections (left panel) ascompared to healthy human brain sections (right panel);

FIG. 6 shows that serum from Rhesus macaques immunized with a conjugate(Conjugate A) according to an embodiment of the invention formulated ina composition containing soluble CpG and alum hydroxide binds topathological tau structures in human AD brain sections (top row), ascompared to healthy human brain sections (bottom row);

FIGS. 7A, 7B, 7C, and 7D show the titers of anti-phosphorylated tauantibodies in Rhesus macaques induced by liposomal vaccines according toembodiments of the invention, Liposomes X, Y and Z, each of whichcontains encapsulated T-cell epitope T50 and one or more adjuvants; thetiters were measured by ELISA and presented in end point titers overtime in individual monkeys. In particular:

FIG. 7A shows the titers of anti-phosphorylated tau antibodies inducedby Liposome X with a TLR4 ligand, MPLA (3D-(6-acyl) PHAD®) alone as theadjuvant;

FIG. 7B shows the titers of anti-phosphorylated tau antibodies inducedby Liposome Y with a TLR9 ligand (lipidated CpG oligonucleotide) aloneas the adjuvant;

FIG. 7C shows the titers of anti-phosphorylated tau antibodies inducedby Liposome Z with a combination of a TLR4 ligand, MPLA (3D-(6-acyl)PHAD®) and a TLR9 ligand (lipidated CpG oligonucleotide) as theadjuvants, which also shows that the combination of two adjuvantsinduces less variability in antibody titers among individual monkeys;

FIG. 7D presents the geometric mean of antibody titers of theabove-mentioned immunization groups and a control liposomal vaccine witha TLR4 ligand, MPLA, but without T-cell epitope, and shows that thevaccines according to embodiments of the invention result in higherantibody titers of anti-phosphorylated tau antibodies than a controlliposomal vaccine: titers were measured by ELISA and are presented ingeometric mean+/−95% confidence interval of end point titers per groupover time;

FIG. 8 shows that immunization using a liposomal vaccine (e.g., LiposomeX, Y or Z) or a conjugate vaccine (Conjugate A) according to anembodiment of the invention induces antibody IgG titers specific forenriched paired helical filaments (ePHF) isolated from the post mortembrain of Alzheimer's disease patients: antibody titers were measured byMeso Scale Discovery (MSD) technology and are presented as values forindividual monkeys on Day 50 and the geometric mean+/−95% CI after thefirst immunization;

FIGS. 9A and 9B show that immunization with a liposomal vaccineaccording to an embodiment of the invention (Liposome Z) containingencapsulated T50 and a combination of a TLR4 ligand (3D-(6-acyl) PHAD®)and a TLR9 ligand (lipidated CpG oligonucleotide) as adjuvants inducesantibodies that mostly bind the N-terminus of phosphorylated tau peptideof SEQ ID NO: 2 (FIG. 9A), whereas monkeys immunized with a conjugatevaccine according to an embodiment of the invention (Conjugate A)generate IgG antibodies that bind mostly to the C-terminal part of thepeptide, for both phosphorylated peptide (left) and non-phosphorylatedpeptide (right) (FIG. 9B);

FIGS. 10A and 10B show that vaccination with a liposomal vaccineaccording to an embodiment of the invention containing encapsulatedT-cell epitope T50 and TLR4 ligand (3D-(6-acyl) PHAD®) as adjuvant(Liposome S) induces significantly higher antibody titers than thecontrol liposomal vaccine (with a TLR4 ligand, 3D-(6-acyl) PHAD®, butwithout T-cell epitope T50, Liposome R) and also the liposomal vaccineaccording to an embodiment of the invention containing surface T-cellepitope T57 (dipalmitoylated T50) and TLR4 ligand (3D-(6-acyl) PHAD®,Liposome T) in mice: antibody titers at day 21 (FIG. 10A) and 35 (FIG.10B) after the first immunization were measured by ELISA and presentedas individual values and the geometric mean per group ±95% CI; (**:p<0.01, ***: p<0.001);

FIGS. 11A and 11B show that encapsulation of T-cell peptides T48 or T52into the liposome (Liposome M or N, respectively) induces a T-cellresponse specific to the encapsulated peptide in mice: T-cell responsewas evaluated by IFN-γ (FIG. 11A) and IL-4 (FIG. 11B) ELISPOT;

FIG. 12 shows that liposomal vaccine containing an encapsulated T-cellepitope (Liposome L) and liposomal vaccine containing an anchored T-cellepitope (Liposome O) each induced higher tau phosphopeptide-specificantibody titers than the control liposomal vaccine without T-cellepitope; each of Liposome L, Liposome O and control liposome furthercontains MPLA as adjuvant;

FIGS. 13A, 13B, 13C, 13D, 13E, 13F, 13G, and 13H show that Tau conjugate(KLH-TAUVAC-p7.1 or KLH-TAUVAC-p22.1) induces TfH cells and robust Abtiters against tau peptide in wild-type mice, in particular:

FIG. 13A illustrates that groups of adult female Balb/C mice (n=14 pergroup) were immunized a total of four times with 100 ug adjuvantedKLH-tau conjugate vaccine (KLH-TAUVAC-p7.1 or KLH-TAUVAC-p22.1), anactive placebo vaccine (KLH plus alum or Ribi), or an inactive placebo(PBS) according to the schedule shown: four animals from eachimmunization group were sacrificed seven days after the primaryimmunization, and the lymph nodes draining the injection site werecollected;

FIG. 13B shows the geometric mean percent of TfHs by immunization group(n=4 mice per group analyzed individually) in the draining nodes: allgroups receiving active vaccines or placebos had measurable TfHs;moreover, animals receiving vaccine KLH-TAUVAC-p7.1, KLH-TAUVAC-p22.1 oractive placebo KLH plus alum, had significantly more TfHs than theanimals given an inactive placebo (p=0.0044 for KLH-TAUVAC-p7.1;p=0.0482 for KLH-TAUVAC-p22.1; p=0.0063 for KLH, using an ANOVA testfollowed by a Dunnett's adjustment for multiple comparisons);

FIGS. 13C, 13D, 13E, 13F, 13G, and 13H show change in serum titers frombaseline (day 0) at four timepoints after immunization (days 14, 28, 56,and 84) for the group means (n=5-10) with 95% confidence interval:asterisk indicates time points at which KLH-TAUVAC-induced antibodyresponse is significantly higher than the one induced by active placebos(p0.05, measured using ANOVA test followed by Tukey's adjustment formultiple comparisons), more specifically:

FIG. 13C shows binding titers to the phosphorylated tau peptide p7.1;

FIG. 13D shows binding titers to the phosphorylated tau peptide p22.1;

FIG. 13E shows binding titers to the non-phosphorylated tau peptide 7.1;

FIG. 13F shows binding titers to the non-phosphorylated tau peptide22.1; and

FIGS. 13G and 13H each show binding titers to the carrier protein KLH;

FIG. 14 shows that sera from mice immunized with Tau conjugate alsobound pathological tau structures from other tauopathies: pooled sera(n=6) from each vaccination group 84 days after primary immunizationwere used to stain brain tissue from a frontal temporal dementia casewith a MAPT mutation (MAPT P301S, frontal cortex), a case with Pick'sdisease (frontal cortex), progressive supranuclear palsy (PSP, caudatenucleus) and primary age-related tauopathy (PART, hippocampus); serafrom animals receiving the active vaccines highlighted the tau-relatedstructures typical of each tauopathy, while sera from animals immunizedwith an active placebo (KLH-alum or KLH-Ribi) or the inactive one (PBS),did not stain any of those structures; as reference an immunostainingwith AT8 of the corresponding area is shown; scale bar=50 um;

FIGS. 15A, 15B, and 15C show that vaccine-induced antibodies reduceaggregated tau in an accelerated tauopathy model, in particular:

FIG. 15A: three month old P301L transgenic mice (n=15 per group)received a stereotactic injection of human ePHF pre-incubated withpurified IgG from mice immunized with either KLH-TAUVAC-p7.1 plus RIBIor with the active placebo KLH plus RIBI; two months after theinjection, all mice were sacrificed and the amount of aggregated tau inthe mice was determined in total and sarkosyl-insoluble fractions;

FIGS. 15B and 15C: the total fractions (FIG. 15B) and sarkosyl-insolublefractions (FIG. 15C) collected from the injected hemisphere of eachanimal: graphs show the amount of tau measured by MSD; in both the totalfraction and the insoluble fraction, brains of mice receiving ePHFpre-incubated with IgG from KLH-TAUVAC-p7.1 immunized mice hadsignificantly less aggregated tau than did mice receiving ePHFpre-incubated with control antibodies. (p<0.0001, using an ANOVA testfollowed by Holm-Bonferroni adjustment for multiple comparisons); and

FIGS. 16A, 16B, 16C, 16D, and 16E show that Tau conjugate (Conjugate B)according to an embodiment of the invention induces high titers ofantibodies against phosphorylated Tau and ePHF in non-human primates:Rhesus macaques were immunized with alum and CpG adjuvantedKLH-TAUVAC-p7.1 (n=6) or with KLH (n=2) at day 1, 29, 85 and 169; bloodwas collected every 14 days, in particular:

FIG. 16A: sera from animals immunized with KLH-TAUVAC-p7.1 were testedfor reactivity on the immunizing peptide p7.1 using ELISA;

FIG. 16B: sera collected from all animals 50 days following primaryimmunization had measurable antibody levels against human ePHF usingMSD, with 3 out of 6 animals showing high reactivity on this antigen;

FIG. 16C: sera collected from animals 50 days following primaryimmunization were applied to human brain sections from healthyindividuals or from AD patients, post-immune sera from KLH-TAUVAC-p7.1group stained pathological tau structures, namely neurofibrillarytangles, neuropil threads and neuritic plaques in AD brain tissue, whilesera from KLH-immunized mice did not show any reactivity, and nostaining was observed on control tissue;

FIG. 16D: when tested in the tau immunodepletion assay, animalsreceiving KLH-TAUVAC-p7.1 had antibodies able to bind and deplete tauseed (p=0.03 at day 50 using an ANOVA test followed by Dunnett'sadjustment for multiple comparisons), while immunization with KLH didnot trigger such antibodies;

FIG. 16E: pre- and post-immunization sera were also tested in theneutralization assay as serially diluted individual samples; changesfrom baseline (CFB) were calculated as difference between FRET countsfor readings at day −14 prior to vaccination (baseline) and postvaccination days 50, 106 and 190 respectively. Response at a specificpost vaccination day (dayi) was then computed as follows: Response=%FRET_day_(i)−% FRET_baseline; a general linear mixed model onaforementioned responses, with animal as random effect, was applied withvariables vaccine groups, day and serum levels treated as categoricalvariables and all their interactions;

FIGS. 17A and 17B show that mice immunized with a conjugate vaccine(Conjugate A) according to an embodiment of the invention and acombination with alum hydroxide (alum) and oligo CpG (CpG) adjuvantresults in higher titer antibody responses to the vaccine peptide: adultfemale C57BL/6 mice (n=5-6 per group) were immunized intramuscularlywith either 2 ug or 0.2 ug of the Conjugate A vaccine, and the conjugatevaccine was either administered alone, with alum, with CpG, or with alumand CpG combined; all mice received a primary immunization on day 0 ofthe study followed by a single booster immunization on day 28; doses forthe alum adjuvant was 500 ug per mouse per injection, and doses for theCpG adjuvant was 20 ug/mouse per injection; the graphs show the resultsof binding ELISA using serum collected from immunized mice with vaccinepeptide T3.5 as the coating antigen, with T3.5 specific mean endpointtiters per group plotted, before immunization (day 0) and at two timepoints after immunization (day 28 and 42), and with error barsrepresenting standard error; the tables show the statistical analysis ofthe results, in which antibody titers were compared using thenon-parametric Kruskal-Wallis Test, and pairwise group comparisons wereassessed using the Wilcoxon Signed Rank test as post-hoc to the KruskalWallis test; in particular:

FIG. 17A: mice were immunized with 2 ug of the Conjugate A vaccine; and

FIG. 17B: mice were immunized with 0.2 ug of the Conjugate A vaccine;and

FIG. 18 shows that tau vaccines according to embodiments of theinvention with different ratios of Tau peptide to T-cell epitope inducesustained high titer anti-phosphorylated tau antibodies in Rhesusmacaques.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise.

Unless otherwise stated, any numerical values, such as a concentrationor a concentration range described herein, are to be understood as beingmodified in all instances by the term “about.” Thus, a numerical valuetypically includes ±10% of the recited value. For example, aconcentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, aconcentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).As used herein, the use of a numerical range expressly includes allpossible subranges, all individual numerical values within that range,including integers within such ranges and fractions of the values unlessthe context clearly indicates otherwise.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the invention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers and are intended to be non-exclusive or open-ended.For example, a composition, a mixture, a process, a method, an article,or an apparatus that comprises a list of elements is not necessarilylimited to only those elements but can include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

It should also be understood that the terms “about,” “approximately,”“generally,” “substantially” and like terms, used herein when referringto a dimension or characteristic of a component of the preferredinvention, indicate that the described dimension/characteristic is not astrict boundary or parameter and does not exclude minor variationstherefrom that are functionally the same or similar, as would beunderstood by one having ordinary skill in the art. At a minimum, suchreferences that include a numerical parameter would include variationsthat, using mathematical and industrial principles accepted in the art(e.g., rounding, measurement or other systematic errors, manufacturingtolerances, etc.), would not vary the least significant digit.

As used herein, the term “tau” or “tau protein”, also known asmicrotubule-associated protein tau, MAPT, neurofibrillary tangleprotein, paired helical filament-tau, PHF-tau, MAPTL, MTBT1, refers toan abundant central and peripheral nervous system protein havingmultiple isoforms. In the human central nervous system (CNS), six majortau isoforms ranging in size from 352 to 441 amino acids in length existdue to alternative splicing (Hanger et al., Trends Mol Med. 15:112-9,2009). Examples of tau include, but are not limited to, tau isoforms inthe CNS, such as the 441-amino acid longest tau isoform (4R2N) that hasfour repeats and two inserts and the 352-amino acid long shortest(fetal) isoform (3RON) that has three repeats and no inserts. Examplesof tau also include the “big tau” isoform expressed in peripheral nervesthat contains 300 additional residues (exon 4a). Friedhoff et al.,Biochimica et Biophysica Acta 1502 (2000) 122-132. Examples of tauinclude a human big tau that is a 758 amino acid-long protein encoded byan mRNA transcript 6762 nucleotides long (NM_016835.4), or isoformsthereof. The amino acid sequence of the exemplified human big tau isrepresented in GenBank Accession No. NP_058519.3. As used herein, theterm “tau” includes homologs of tau from species other than human, suchas Macaca Fascicularis (cynomolgous monkey) or Pan troglodytes(chimpanzee). As used herein, the term “tau” includes proteinscomprising mutations, e.g., point mutations, fragments, insertions,deletions and splice variants of full length wild type tau. The term“tau” also encompasses post-translational modifications of the tau aminoacid sequence. Post-translational modifications include, but are notlimited to, phosphorylation.

As used herein, the term “peptide” or “polypeptide” refers to a polymercomposed of amino acid residues, related naturally occurring structuralvariants, and synthetic non-naturally occurring analogs thereof linkedvia peptide bonds. The term refers to a peptide of any size, structure,or function. Typically, a peptide is at least three amino acids long. Apeptide can be naturally occurring, recombinant, or synthetic, or anycombination thereof. Synthetic peptides can be synthesized, for example,using an automated polypeptide synthesizer. Examples of tau peptidesinclude any peptide of tau protein of about 5 to about 30 amino acids inlength, preferably of about 10 to about 25 amino acids in length, morepreferably of about 16 to about 21 amino acids in length. In the presentdisclosure, peptides are listed from N to C terminus using the standardthree or one letter amino acid abbreviation, wherein phosphoresidues areindicated with “p”. Examples of tau peptides useful in the inventioninclude, but are not limited to, tau peptides comprising the amino acidsequence of any of SEQ ID NOs: 1-12, or tau peptides having an aminoacid sequence that is at least 75%, 80%, 85%, 90% or 95% identical tothe amino acid sequence of any of SEQ ID NOs: 1-12.

As used herein, the term “phosphopeptide” or “phospho-epitope” refers toa peptide that is phosphorylated at one or more amino acid residues.Examples of tau phosphopeptides include any tau peptide comprising oneor more phosphorylated amino acid residue. Examples of tauphosphopeptides useful in the invention include, but are not limited to,tau phosphopeptides comprising the amino acid sequence of any of SEQ IDNOs: 1-3 or 5-12, or tau phosphopeptides having an amino acid sequencethat is at least 75%, 80%, 85%, 90% or 95% identical to the amino acidsequence of any of SEQ ID NOs: 1-3 or 5-12.

The tau peptides of the present invention can be synthesized by solidphase peptide synthesis or by recombinant expression systems. Automaticpeptide synthesizers are commercially available from numerous suppliers,such as Applied Biosystems (Foster City, Calif.). Recombinant expressionsystems can include bacteria, such as E. coli, yeast, insect cells, ormammalian cells. Procedures for recombinant expression are described bySambrook et al., Molecular Cloning: A Laboratory Manual (C.S.H.P. Press,NY 2d ed., 1989).

Tau is a human “self” protein. This means that, in principle, alllymphocytes bearing a receptor specific for tau should have been deletedduring development (central tolerance) or rendered unresponsive by aperipheral tolerance mechanism. This problem has proved to be asignificant roadblock to the development of vaccines against self or“altered self” proteins (e.g. tumor antigens).

Generating high-quality antibodies against an antigen (self orinfectious) requires the action of not only B lymphocytes, which producethe antibody, but also of CD4+ T “helper” lymphocytes. CD4+ T-cellsprovide critical survival and maturation signals to B lymphocytes, andCD4+ T-cell deficient animals are profoundly immunosuppressed. CD4+T-cells are also subject to tolerance mechanisms, and an additionalroadblock to generating strong anti-self (e.g., anti-tau) antibodyresponses is that tau-reactive CD4+ T-cells are also likely to be rareto non-existent in the human/animal repertoire.

While not wishing to be bound by theory, it is believed, but in no waylimiting the scope of the present invention, that this problem iscircumvented by vaccine compositions of the present invention.

In one embodiment, a liposome comprising a tau peptide (one example isshown in FIG. 1; top) is produced that also comprises a T-cell epitopethat is capable of binding most or all HLA DR (Human LeukocyteAntigen—antigen D Related) molecules. The T-cell epitope is then able toactivate CD4+ T-cells and provides essential maturation and survivalsignals to the tau-specific B-cells (FIG. 2). In another embodiment, aconjugate of a tau peptide with a carrier protein is produced (oneexample is shown in FIG. 1; bottom), which generates a strong helperT-cell response (FIG. 3). In this embodiment “non-linked recognition” isused, in which carrier-specific T-cells provide survival and maturationsignals to self-reactive B-cells. Accordingly, the tau-specific B-cellsreceive crucial signals to trigger affinity maturation, immunoglobulinclass switching, and to establish a long-term memory pool. The tauliposomes and tau conjugates can be used to generate high-qualityantibodies against the tau antigen in homologous or heterologousimmunization schemes, with either liposome or conjugate used in theprime and/or in the boost.

Liposomes

In one general aspect, the invention relates to a liposome, comprising:

a. a tau peptide, preferably the tau peptide is a tau phosphopeptide;and

b. a helper T-cell epitope,

wherein the tau peptide is presented on the surface of the liposome.

Liposomes according to embodiments of the invention are also referred toherein as “improved liposomes,” “improved liposomal vaccines” or“liposomal vaccines according to embodiments of the invention” or “Tauliposomes” or “optimized liposomal vaccines” of “2^(nd) generationliposomes”.

As used herein, the term “liposome” refers generally to a lipid vesiclethat is made of materials having high lipid content, e.g.,phospholipids, cholesterol. The lipids of these vesicles are generallyorganized in the form of lipid bilayers. The lipid bilayers generallyencapsulate a volume which is either interspersed between multipleonion-like shells of lipid bilayers, forming multilamellar lipidvesicles (MLVs) or contained within an amorphous central cavity. Lipidvesicles having an amorphous central cavity are unilamellar lipidvesicles, i.e., those with a single peripheral bilayer surrounding thecavity. Large unilamellar vesicles (LUVs) generally have a diameter of100 nm to few micrometer, such as 100-200 nm or larger, while smallunilamellar lipid vesicles (SUV) generally have a diameter of less than100 nm, such as 20-100 nm, typically 15-30 mm.

According to particular embodiments, the liposome comprises one or moretau peptides. According to particular embodiments, the tau peptides inthe liposome can be the same or different.

Any suitable tau peptide known to those skilled in the art can be usedin the invention in view of the present disclosure. According toparticular embodiments, one or more of the tau peptides comprise theamino acid sequence of one of SEQ ID NOs: 1-12. In other embodiments,one or more of the tau peptides comprise an amino acid sequence that isat least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequenceof one of SEQ ID NOs: 1-12, wherein none of the amino acid residues arephosphorylated, or one or more amino acid residues are phosphorylated.

According to particular embodiments, one or more of the tau peptides isa tau phosphopeptide. According to particular embodiments, the one ormore tau phosphopeptides comprise the amino acid sequence of one of SEQID NOs: 1-3 or 5-12, or an amino acid sequence that is at least 75%,80%, 85%, 90% or 95% identical to the amino acid sequence of one of SEQID NOs: 1-3 or 5-12, wherein one or more of the indicated amino acidresidues are phosphorylated. Preferably, the tau phosphopeptidecomprises the amino acid sequence of one of SEQ ID Nos: 1-3. The taupeptide can have the C-terminus amidated.

According to embodiments of the application, a tau peptide is presentedon the surface of the liposome. A tau peptide, preferably a tauphosphopeptide, can be presented on the surface of the liposome usingmethods known in the art in view of the present disclosure. See, forexample, the relevant disclosure in U.S. Pat. Nos. 8,647,631 and9,687,447, the content of which is incorporated herein by reference.According to particular embodiments, the one or more tau peptides,including phosphopeptides, further comprise one or more modifications,such as palmitoylation or dodecyl modification to allow the tau peptidesto be presented on the surface of the liposome. Additional amino acidresidues, such as Lys, Cys, or sometimes Ser or Thr, can be added to thetau peptide to facilitate the modification. It was reported that theposition of lipid anchors induces different conformations of the peptidesequence (Hickman et al., J. Biol. Chem. vol. 286, NO. 16, pp.13966-13976, Apr. 22, 2011). While not wishing to be bound by theory, itis believed that adding hydrophobic moieties at both termini mayincrease the pathological beta-sheet conformation of the tau peptide.Thus, the one or more tau peptides further comprise hydrophobic moietiesat both termini. The modified tau peptide can have the C-terminusamidated. Preferably, a tau peptide presented on the surface of theliposome consists of the amino acid sequence of one of SEQ ID NO:27 toSEQ ID NO:38.

As used herein, the term “helper T-cell epitope” refers to a polypeptidecomprising an epitope that is capable of recognition by a helper T-cell.Examples of helper T-cell epitopes include, but are not limited to,tetanus toxoid (e.g., the P2 and P30 epitopes, also named, respectivelyas T2 and T30), Hepatitis B surface antigen, cholera toxin B, toxoid,diphtheria toxoid, measles virus F protein, Chlamydia trachomatis majorouter membrane protein, Plasmodium falciparum circumsporozite T, P.falciparum CS antigen, Schistosoma mansoni triose phosphate isomerase,Bordetella pertussis, Clostridium tetani, Pertusaria trachythallina,Escherichia coli TraT, and Influenza virus hemagglutinin (HA).

Any suitable helper T-cell epitope known to those skilled in the art canbe used in the invention in view of the present disclosure. According toparticular embodiments, the helper T-cell epitope comprises at least oneamino acid sequence selected from the group consisting of SEQ ID NO:23to SEQ ID NO:26. Preferably, the helper T-cell epitope comprises two ormore of the amino acid sequences of SEQ ID NO:23 to SEQ ID NO:26 fusedtogether via a linker, such as a peptide linker comprising one or moreamino acids, e.g., Val (V), Ala (A), Arg (R), Gly (G), Ser (S), Lys (K).The length of the linker can vary, preferably 1-5 amino acids.Preferably, the helper T-cell epitope comprises three or more of theamino acid sequences of SEQ ID NO:23 to SEQ ID NO:26 fused together viaone or more linkers selected from the group consisting of VVR, GS, RR,RK. The helper T-cell epitope can have its C-terminus amidated.

According to embodiments of the application, the helper T-cell epitopescan be incorporated on the liposomal surface, e.g. anchored by acovalently bound hydrophobic moiety wherein said hydrophobic moiety isan alkyl group, a fatty acid, a triglyceride, diglyceride, steroid,sphingolipid, glycolipid or a phospholipid, particularly an alkyl groupor a fatty acid, particularly with a carbon backbone of at least 3carbon atoms, particularly of at least 4 carbon atoms, particularly ofat least 6 carbon atoms, particularly of at least 8 carbon atoms,particularly of at least 12 carbon atoms, particularly of at least 16carbon atoms. In one embodiment of the invention, the hydrophobic moietyis palmitic acid. Alternatively, the helper T-cell epitopes can beencapsulated in the liposomes. According to particular embodiments, thehelper T-cell epitope is encapsulated in the liposome.

The helper T-cell epitope can be modified for its desired location inthe liposomes using methods known in the art in view of the presentdisclosure. According to particular embodiments, the helper T-cellepitope useful for the invention comprises an amino acid sequence of oneof SEQ ID NO:39 to SEQ ID NO:44. Preferably, the helper T cell epitopeconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO:13 to SEQ ID NO:17.

According to particular embodiments, the liposome comprises a taupeptide and a helper T-cell epitope ata weight ratio of 1:1, 2:1, 3:1,4:1, 5:1 or 6:1.

In an embodiment, the liposome further comprises at least one adjuvantcomprising a toll-like receptor ligand. Thus, in another general aspect,the invention relates to a liposome, comprising:

a. a tau peptide, preferably a tau phosphopeptide;

b. a helper T-cell epitope; and

c. at least one of

-   -   i. a toll-like receptor 9 ligand, and    -   ii. a toll-like receptor 4 ligand.

As used herein, the term “toll-like receptor” or “TLR” refers to a classof pattern recognition receptor (PRR) proteins that play a key role inthe innate immune response. TLRs recognize pathogen-associated molecularpatterns (PAMPs) from microbial pathogens, such as bacteria, fungi,parasites and viruses, which can be distinguished from host molecules.TLRs are membrane-spanning proteins that typically function as dimersand are expressed by cells involved in the innate immune response,including antigen-presenting dendritic cells and phagocytic macrophages.There are at least ten human TLR family members, TLR1 to TLR10, and atleast twelve murine TLR family members, TLR1 to TLR9 and TLR11 to TLR13,and they differ in the types of antigens they recognize. For example,TLR4 recognizes lipopolysaccharides (LPS), a component present in manyGram-negative bacteria, as well as viral proteins, polysaccharide, andendogenous proteins such as low-density lipoprotein, beta-defensins andheat shock protein; and TLR9 is a nucleotide-sensing TLR which isactivated by unmethylated cytosine-phosphate-guanine (CpG)single-stranded or double-stranded dinucleotides, which are abundant inprokaryotic genomes but rare in vertebrate genomes. Activation of TLRsleads to a series of signaling events resulting in the production oftype I interferons (IFNs), inflammatory cytokines, and chemokines, andthe induction of immune responses. Eventually, this inflammation alsoactivates the adaptive immune system, which then results in theclearance of the invading pathogens and the infected cells.

As used herein, the term “ligand” refers to a molecule that forms acomplex with a biomolecule (e.g., a receptor) to serve a biologicalpurpose. According to particular embodiments, the toll-like receptorligand is a toll-like receptor agonist.

As used herein, the term “agonist” refers to a molecule that binds toone or more TLRs and induces a receptor mediated response. For example,an agonist can induce, stimulate, increase, activate, facilitate,enhance, or up regulate the activity of the receptor. Such activitiesare referred to as “agonistic activities.” For example, a TLR4 or TLR9agonist can activate or increase cell signaling through the boundreceptor. Agonists include, but are not limited to nucleic acids, smallmolecules, proteins, carbohydrates, lipids or any other molecules thatbind or interact with receptors. Agonists can mimic the activity of anatural receptor ligand. Agonists can be homologous to these naturalreceptor ligands with respect to sequence, conformation, charge or othercharacteristics such that they can be recognized by the receptors. Thisrecognition can result in physiologic and/or biochemical changes withinthe cell, such that the cell reacts to the presence of the agonist inthe same manner as if the natural receptor ligand were present.According to particular embodiments, the toll-like receptor agonist isat least one of a toll-like receptor 4 agonist and a toll-like receptor9 agonist.

As used herein, the term “toll-like receptor 4 agonist” refers to anycompound that acts as an agonist of TLR4. Any suitable toll-likereceptor 4 agonist known to those skilled in the art in view of thepresent disclosure can be used in the invention. Examples of toll-likereceptor 4 ligand useful for the invention include TLR4 agonist,including, but not limited to, monophosphoryl lipid A (MPLA). As usedherein, the term “monophosphoryl lipid A” or MPLA″ refers to a modifiedform of lipid A, which is the biologically active part of Gram-negativebacterial lipopolysaccharide (LPS) endotoxin. MPLA is less toxic thanLPS while maintaining the immunostimulatory activity. As a vaccineadjuvant, MPLA stimulates both cellular and humoral responses to thevaccine antigen. Examples of MPLA include, but are not limited to,3-O-desacyl-4′-monophosphoryl lipid A, monophosphoryl hexa-acyl lipid A,3-deacyl, monophosphoryl 3-deacyl lipid A, and structurally relatedvariants thereof. MPLA useful for the invention can be obtained usingmethods known in the art, or from a commercial source, such as3D-(6-acyl) PHAD®, PHAD®, PHAD®-504, 3D-PHAD® from Avanti Polar Lipids(Alabaster, Ala., USA) or MPLTM from various commercial sources.According to particular embodiments, the toll-like receptor 4 agonist isMPLA.

As used herein, the term “toll-like receptor 9 agonist” refers to anycompound that acts as an agonist of TLR9. Any suitable toll-likereceptor 9 agonist known to those skilled in the art in view of thepresent disclosure can be used in the invention. Examples of toll-likereceptor 9 ligand useful for the invention include TLR9 agonistincluding, but not limited to, CpG oligonucleotides.

As used herein, the term “CpG oligonucleotide”, “CpGoligodeoxynucleotide” or “CpG ODN” refers to an oligonucleotidecomprising at least one CpG motif. As used herein, “oligonucleotide,”“oligodeoxynucleotide” or “ODN” refers to a polynucleotide formed from aplurality of linked nucleotide units. Such oligonucleotides can beobtained from existing nucleic acid sources or can be produced bysynthetic methods. As used herein, the term “CpG motif” refers to anucleotide sequence which contains unmethylatedcytosine-phosphate-guanine (CpG) dinucleotides (i.e., a cytosine (C)followed by a guanine (G)) linked by a phosphate bond or aphosphodiester backbone or other internucleotide linkages.

According to particular embodiments, the CpG oligonucleotide islipidated, i.e. conjugated (covalently linked) to a lipid moiety.

As used herein, a “lipid moiety” refers to a moiety containing alipophilic structure. Lipid moieties, such as an alkyl group, a fattyacid, a triglyceride, diglyceride, steroid, sphingolipid, glycolipid ora phospholipid, particularly a sterol such as cholesterol, or fattyacids, when attached to highly hydrophilic molecules, such as nucleicacids, can substantially enhance plasma protein binding and consequentlycirculation half-life of the hydrophilic molecules. In addition, bindingto certain plasma proteins, such as lipoproteins, has been shown toincrease uptake in specific tissues expressing the correspondinglipoprotein receptors (e.g., LDL-receptor HDL-receptor or the scavengerreceptor SR-B1). In particular, a lipid moiety conjugated to thephosphopeptides and/or CpG oligonucleotide allows anchoring the saidpeptides and/or oligonucleotides into the membrane of a liposome via ahydrophobic moiety.

According to particular embodiments, in view of the present disclosure,the CpG oligonucleotide can comprise any suitable internucleotidelinkages.

As used herein, the term “internucleotide linkage” refers to a chemicallinkage to join two nucleotides through their sugars consisting of aphosphorous atom and a charged or neutral group between adjacentnucleosides. Examples of internucleotide linkage include phosphodiester(po), phosphorothioate (ps), phosphorodithioate (ps2), methylphosphonate(mp), and methylphosphorothioate (rp). Phosphorothioate,phosphorodithioate, methylphosphonate and methylphosphorothioate arestabilizing internucleotide linkages, while phosphodiester is anaturally-occurring internucleotide linkage. Oligonucleotidephosphorothioates are typically synthesized as a random racemic mixtureof Rp and Sp phosphorothioate linkages.

Any suitable CpG oligonucleotide known to those skilled in the art canbe used in the invention in view of the present disclosure. Examples ofsuch CpG oligonucleotides include, but are not limited to CpG2006 (alsoknown as CpG 7909), CpG 1018, CpG2395, CpG2216 or CpG2336.

A CpG oligonucleotide can be lipidated using methods known in the art inview of the present disclosure. In some embodiments, 3′ terminus of aCpG oligonucleotide is covalently linked to a cholesterol moleculethrough a phosphate bond, optionally via a PEG linker. Other lipophilicmoiety can also be covalently linked to the 3′ terminus of a CpGoligonucleotide. For example a CpG oligonucleotide can be covalentlylinked to a lipid anchor of the same length as the phospholipids fromliposome: one palmitic acid chain (using Pal-OH or similar, activatedfor coupling) or two palmitic acids (e.g., using1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) orsimilar, activated for coupling), optionally via a PEG linker. See,e.g., relevant disclosure in U.S. Pat. No. 7,741,297, the content ofwhich is incorporated herein by reference. The length of PEG can vary,from example, from 1 to 5 PEG units.

Other linkers can also be used to covalently connect a CpGoligonucleotide to a lipophilic moiety (such as a cholesterol molecule),examples of which include, but are not limited to an alkyl spacer having3 to 12 carbons. A short linker compatible with oligonucleotidechemistry is needed as aminodiol. In some embodiment, no linker is usedfor the covalent bonding. See e.g., Ries et al., “Convenient synthesisand application of versatile nucleic acid lipid membrane anchors in theassembly and fusion of liposomes, Org. Biomol. Chem., 2015, 13, 9673,the relevant disclosure of which is incorporated herein by reference.

According to particular embodiments, lipidated CpG oligonucleotideuseful for the invention comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NO:18 to SEQ ID NO:22, wherein thenucleotide sequence comprises one or more phosphorothioateinternucleotide linkages, and the nucleotide sequence is covalentlylinked to at least one cholesterol via a linker. Any suitable linkerscan be used to covalently link a CpG oligonucleotide to a cholesterolmolecule. Preferably, the linker comprises polyethylene glycol (PEG).

According to particular embodiments, the liposome comprises:

a. a tau phosphopeptide;

b. a helper T-cell epitope;

c. a lipidated CpG oligonucleotide; and

d. a toll-like receptor 4 ligand;

wherein the tau phosphopeptide is presented on the surface of theliposome, and the helper T-cell epitope is encapsulated in the liposome.

According to particular embodiments, the liposome comprises:

-   -   a. a tau peptide having an amino acid sequence selected from the        group consisting of SEQ ID NO:27 to SEQ ID NO:38;    -   b. a helper T cell epitope having an amino acid sequence        selected from the group consisting of SEQ ID NO:39 to SEQ ID        NO:44, preferably, the helper T cell epitope consisting of an        amino acid sequence selected from the group consisting of SEQ ID        NO:13 to SEQ ID NO:17;    -   c. a lipidated CpG oligonucleotide having a nucleotide sequence        selected from the group consisting of SEQ ID NO:18 to SEQ ID        NO:22, wherein the CpG oligonucleotide comprises one or more        phosphorothioate internucleotide linkages, and the CpG        oligonucleotide is covalently linked to at least one cholesterol        via a linker; and    -   d. monophosphoryl lipid A (MPLA).

According to particular embodiments, the liposome further comprises oneor more lipids selected from the group consisting of1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dimyristoyl-sn-glycero-3-phosphoryl-3′-rac-glycerol (DMPG), andcholesterol.

According to particular embodiments, the liposome further comprises abuffer. Any suitable buffer known to those skilled in the art in view ofthe present disclosure can be used in the invention. In one embodiment,the liposome comprises a phosphate-buffered saline. According toparticular embodiments, the buffer comprises histidine and sucrose.

According to particular embodiments, the liposome comprises DMPC, DMPG,cholesterol, tau phosphopeptide and helper T-cell epitope at a molarratio of 9:1:7:0.07:0.04.

Liposomes of the invention can be made using methods known in the art inview of the present disclosure. 1001.061 An exemplary liposome of thepresent application is illustrated in FIG. 1. More specifically, a tautetrapalmitoylated phosphopeptide (pTau Peptide T3, SEQ ID NO: 28) ispresented on the surface of the liposome via two palmitic acids at eachterminus of the tau peptide. A TLR-9 ligand comprising lipidated CpG(Adjuvant CpG7909-Chol) is incorporated into the liposome membrane viathe covalently linked cholesterol. A TLR-4 ligand (Adjuvant 3D-(6-acyl)PHAD®) is also incorporated into the membrane. A helper T-cell epitope(PAN-DR binder T50) is encapsulated.

Conjugates

In one general aspect, the invention relates to a conjugate comprising atau peptide and an immunogenic carrier conjugated thereto.

According to particular aspects, the conjugate has the followingstructure:

or the structure of formula (II):

wherein

x is an integer of 0 to 10; and

n is an integer of 2 to 15, preferably 3-11.

According to particular embodiments, x is an integer of 1 to 10, 2 to 9,2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. According toparticular embodiments, x is 3.

According to particular embodiments, n is 2 to 15, 3 to 11, 3 to 9, 3 to8, or 3 to 7.

According to particular embodiments, the conjugate comprises one or moretau peptides. According to particular embodiments, the tau peptides ofthe conjugate can be the same or different.

According to particular embodiments, in view of the present disclosure,any suitable tau peptides can be used in the invention. According toparticular embodiments, one or more of the tau peptides comprise theamino acid sequence of one of SEQ ID NOs: 1-12, or an amino acidsequence that is at least 75%, 80%, 85%, 90% or 95% identical to theamino acid sequence of one of SEQ ID NOs: 1-12, wherein none, one ormore of the amino acid residues are phosphorylated.

According to particular embodiments, one or more of the tau peptides isa tau phosphopeptide. According to particular embodiments, the one ormore tau phosphopeptides comprise the amino acid sequence of one of SEQID NOs: 1-3 or 5-12, or an amino acid sequence that is at least 75%,80%, 85%, 90% or 95% identical to the amino acid sequence of one of SEQID NOs: 1-3 or 5-12, wherein one or more of the indicated amino acidresidues are phosphorylated.

According to particular embodiments, the tau phosphopeptide consists ofthe amino acid sequence of one of SEQ ID NOs: 1-3.

As used herein, the term “immunogenic carrier” refers to an immunogenicsubstance that can be coupled to a tau peptide. An immunogenic moietycoupled to a tau peptide can induce an immune response and elicit theproduction of antibodies that can specifically bind the tau peptide.Immunogenic moieties are operative moieties that include proteins,polypeptides, glycoproteins, complex polysaccharides, particles, nucleicacids, polynucleotides, and the like that are recognized as foreign andthereby elicit an immunologic response from the host. Any suitableimmunogenic carrier known to those skilled in the art in view of thepresent disclosure can be used in the invention. According to particularembodiments, the immunogenic carrier is keyhole limpet hemocyanin (KLH),tetanus toxoid, CRM197 (a non-toxic form of diphtheria toxin), an outermembrane protein mixture from N. meningitidis (OMP), or a derivativethereof. According to particular embodiments, the immunogenic carrier isKLH or CRM197.

According to particular embodiments, the tau peptide is conjugated tothe carrier via a linker. As used herein, the term “linker” refers to achemical moiety that joins a immunogenic carrier to a tau peptide. Anysuitable linker known to those skilled in the art in view of the presentdisclosure can be used in the invention. The linkers can be, forexample, a single covalent bond, a substituted or unsubstituted alkyl, asubstituted or unsubstituted heteroalkyl moiety, a polyethylene glycol(PEG) linker, a peptide linker, a sugar-based linker, or a cleavablelinker, such as a disulfide linkage or a protease cleavage site, or anamino acid, or a combination thereof. Examples of the linker cancomprises one or more of polyethylene glycol (PEG), succinimidyl3-(bromoacetamido)propionate (SBAP),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), or one or moreamino acids such as Cys, Lys or sometimes Ser or Thr, or a combinationthereof.

According to particular embodiments, the linker comprises(C2H4O)x-cysteine-acetamidopropionamide orm-maleimidobenzoyl-N-hydroxysuccinimide ester—cysteine—(C2H4O)x, whereinx is an integer of 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

According to particular embodiments, the carrier is covalently linked tothe N-terminus of the tau peptide, via a linker.

According to other particular embodiments, the carrier is covalentlylinked to the C-terminus of the tau peptide, via a linker.

According to particular embodiments, the conjugate has the structure of:

wherein n is an integer of 2 to 15, preferably 3-11, more preferably3-7.

Conjugates of the invention can be made by methods known in the art inview of the present disclosure. For example, the above conjugate can beformed by reacting succinimidyl-3-(bromoacetamido)propionate (SBAP):

with an amino group of CRM197 to form an amide linkage. This CRM197precursor can be subsequently reacted with the tau peptide (e.g., thephosphorylated tau peptide of SEQ ID NO: 2) conjugated at its N-terminusor at its C-terminus to a PEG-cysteine linker with a free nucleophilicthiol group to form the tau phosphopeptide conjugate.

An exemplary conjugate according to an embodiment of the presentapplication is illustrated in FIG. 1. More specifically, multiple tauphosphopeptides (pTau Peptide T3.76) are covalently linked to a carrierprotein CRM197.

Pharmaceutical Compositions

In one general aspect, the invention relates to pharmaceuticalcompositions comprising a therapeutically effective amount of liposomeor conjugate of the invention, together with a pharmaceuticallyacceptable excipient and/or carrier. Pharmaceutically acceptableexcipients and/or carriers are well known in the art (see Remington'sPharmaceutical Science (15th ed.), Mack Publishing Company, Easton, Pa.,1980). The preferred formulation of the pharmaceutical compositiondepends on the intended mode of administration and therapeuticapplication. The compositions can include pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, non-immunogenicstabilizers, and the like. It will be understood that thecharacteristics of the carrier, excipient or diluent will depend on theroute of administration for a particular application.

The pharmaceutical composition can contain a mixture of the sameimmunogenic tau peptide. Alternatively, the pharmaceutical compositioncan contain a mixture of different immunogenic tau peptides of thepresent invention.

Another problem associated with vaccines against neuronal diseases isthat exceptionally high antibody titers are likely to be necessary toassure efficacy. This is because the target antigen for the vaccine islocated in the brain. The brain is separated from the circulation by aspecialized cellular structure called the blood-brain barrier (BBB). TheBBB restricts passage of substances from the circulation into the brain.This prevents the entry of toxins, microbes, etc. into the centralnervous system. The BBB also has the potentially less desirable effectof preventing the efficient entry of immune mediators (such asantibodies) into the interstitial and cerebrospinal fluid that surroundsthe brain.

Approximately 0.1% of antibodies that are present in the systemiccirculation cross the BBB and enter the brain. This means that systemictiters induced by a vaccine targeting a CNS antigen must be at least1000 times greater than the minimal effective titer to be efficacious inthe brain.

According to particular embodiments, the pharmaceutical compositions ofthe present invention therefore further comprise one or more suitableadjuvants. Thus, the tau peptides of the present invention, present inthe liposome or the conjugate, can be administered in combination with asuitable adjuvant to achieve the desired immune response in the subject.Suitable adjuvants can be administered before, after, or concurrent withadministration of liposome or conjugate of the present invention.Preferred adjuvants augment the intrinsic response to an immunogenwithout causing conformational changes in the immunogen that affect thequalitative form of the response. Examples of adjuvants are the aluminumsalts (alum), such as aluminum hydroxide, aluminum phosphate, andaluminum sulfate. Such adjuvants can be used with or without otherspecific immunostimulating agents, such as MPLA Class (3 De-O-acylatedmonophosphoryl lipid A (MPLTM), monophosphoryl hexa-acyl Lipid A3-deacyl synthetic (3D-(6-acyl) PHAD®), PHADTM, PHAD®-504, 3D-PHAD®)lipid A), polymeric or monomeric amino acids, such as polyglutamic acidor polylysine. Such adjuvants can be used with or without other specificimmunostimulating agents, such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) TheramideTM), or other bacterial cell wallcomponents. Oil-in-water emulsions include MF59 (see WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer; SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion; and the RibiTM adjuvant system (RAS)(Ribi ImmunoChem, Hamilton, Mont.) 0.2% Tween 80, and one or morebacterial cell wall components selected from the group consisting ofmonophosphoryl lipid A (MPLTM), trehalose dimycolate (TDM), and cellwall skeleton (CWS), preferably 1MPLTM+CWS (DetoxTM). Other adjuvantsinclude Complete Freund's Adjuvant (CFA), and cytokines, such asinterleukins (IL-1, IL-2, and IL-12), macrophage colony stimulatingfactor (M-CSF), and tumor necrosis factor (TNF).

As used herein, the term “in combination,” in the context of theadministration of two or more therapies to a subject, refers to the useof more than one therapy. The use of the term “in combination” does notrestrict the order in which therapies are administered to a subject. Forexample, a first therapy (e.g., a composition described herein) can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, orsubsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours,72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks,8 weeks, or 12 weeks after) the administration of a second therapy to asubject.

Pharmaceutical compositions of the present invention can be formulatedaccording to methods well known in the art. The optimal ratios of eachcomponent in the compositions can be determined by techniques well knownto those skilled in the art in view of the present disclosure.

Methods of Use

Another general aspect of the invention relates to methods for inducingan immune response against tau protein in a subject suffering from aneurodegenerative disease, disorder, or condition, comprisingadministering to the subject a pharmaceutical composition according toan embodiment of the invention. According to particular aspects, theimmune response is induced against phosphorylated tau protein,preferably ePHF.

Another general aspect of the invention relates to methods for treatingor preventing a neurodegenerative disease, disorder, or condition,comprising administering to the subject a pharmaceutical compositionaccording to an embodiment of the invention.

As used herein, the terms “induce” and “stimulate” and variationsthereof refer to any measurable increase in cellular activity. Inductionof an immune response can include, for example, activation,proliferation, or maturation of a population of immune cells, increasingthe production of a cytokine, and/or another indicator of increasedimmune function. In certain embodiments, induction of an immune responsecan include increasing the proliferation of B cells, producingantigen-specific antibodies, increasing the proliferation ofantigen-specific T cells, improving dendritic cell antigen presentationand/or an increasing expression of certain cytokines, chemokines andco-stimulatory markers.

The ability to induce or stimulate an anti-tau immune response uponadministration in an animal or human organism can be evaluated either invitro or in vivo using a variety of assays which are standard in theart. For a general description of techniques available to evaluate theonset and activation of an immune response, see for example Coligan etal. (1992 and 1994, Current Protocols in Immunology; ed. J Wiley & SonsInc, National Institute of Health). Measurement of cellular immunity canbe performed by methods readily known in the art, e.g., by measurementof cytokine profiles secreted by activated effector cells includingthose derived from CD4+ and CD8+ T-cells (e.g. quantification of IL-4 orIFN gamma-producing cells by ELISPOT), by determination of theactivation status of immune effector cells (e.g. T-cell proliferationassays by a classical [3H] thymidine uptake), by assaying forantigen-specific T lymphocytes in a sensitized subject (e.g.peptide-specific lysis in a cytotoxicity assay, etc.).

The ability to stimulate a cellular and/or a humoral response can bedetermined by testing a biological sample (e.g., blood, plasma, serum,PBMCs, urine, saliva, feces, CSF or lymph fluid) from the subject forthe presence of antibodies directed to the immunogenic tau peptide(s)administered in the pharmaceutical composition (see for example Harlow,1989, Antibodies, Cold Spring Harbor Press). For example, titers ofantibodies produced in response to administration of a compositionproviding an immunogen can be measured by enzyme-linked immunosorbentassay (ELISA), dot blots, SDS-PAGE gels, ELISPOT or Antibody-DependentCellular Phagocytosis (ADCP) Assay.

As used herein, the term “subject” refers to an animal. According toparticular embodiments, the subject is a mammal including a non-primate(e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog,rat, rabbit, guinea pig or mouse) or a primate (e.g., a monkey,chimpanzee or human). According to particular embodiments, the subjectis a human.

As used herein, the term “therapeutically effective amount” refers to anamount of an active ingredient or component that elicits the desiredbiological or medicinal response in a subject. A therapeuticallyeffective amount can be determined empirically and in a routine manner,in relation to the stated purpose. For example, in vitro assays canoptionally be employed to help identify optimal dosage ranges. Selectionof a particular effective dose can be determined (e.g., via clinicaltrials) by those skilled in the art based upon the consideration ofseveral factors, including the disease to be treated or prevented, thesymptoms involved, the patient's body mass, the patient's immune statusand other factors known by the skilled artisan. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the severity of disease, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

As used herein, the terms “treat”, “treating”, and “treatment” are allintended to refer to an amelioration or reversal of at least onemeasurable physical parameter related to a neurodegenerative disease,disorder, or condition, which is not necessarily discernible in thesubject, but can be discernible in the subject. The terms “treat”,“treating”, and “treatment” can also refer to causing regression,preventing the progression, or at least slowing down the progression ofthe disease, disorder, or condition. In a particular embodiment,“treat”, “treating”, and “treatment” refer to an alleviation, preventionof the development or onset, or reduction in the duration of one or moresymptoms associated with the neurodegenerative disease, disorder, orcondition. In a particular embodiment, “treat”, “treating”, and“treatment” refer to prevention of the recurrence of the disease,disorder, or condition. In a particular embodiment, “treat”, “treating”,and “treatment” refer to an increase in the survival of a subject havingthe disease, disorder, or condition. In a particular embodiment,“treat”, “treating”, and “treatment” refer to elimination of thedisease, disorder, or condition in the subject.

According to particular embodiments, a therapeutically effective amountrefers to the amount of therapy which is sufficient to achieve one, two,three, four, or more of the following effects: (i) reduce or amelioratethe severity of the disease, disorder or condition to be treated or asymptom associated therewith; (ii) reduce the duration of the disease,disorder or condition to be treated, or a symptom associated therewith;(iii) prevent the progression of the disease, disorder or condition tobe treated, or a symptom associated therewith; (iv) cause regression ofthe disease, disorder or condition to be treated, or a symptomassociated therewith; (v) prevent the development or onset of thedisease, disorder or condition to be treated, or a symptom associatedtherewith; (vi) prevent the recurrence of the disease, disorder orcondition to be treated, or a symptom associated therewith; (vii) reducehospitalization of a subject having the disease, disorder or conditionto be treated, or a symptom associated therewith; (viii) reducehospitalization length of a subject having the disease, disorder orcondition to be treated, or a symptom associated therewith; (ix)increase the survival of a subject with the disease, disorder orcondition to be treated, or a symptom associated therewith; (x) inhibitor reduce the disease, disorder or condition to be treated, or a symptomassociated therewith in a subject; and/or (xi) enhance or improve theprophylactic or therapeutic effect(s) of another therapy.

As used herein a “neurodegenerative disease, disorder, or condition”includes any neurodegenerative disease, disorder, or condition known tothose skilled in the art in view of the present disclosure. Examples ofneurodegenerative diseases, disorders, or conditions includeneurodegenerative diseases or disorders caused by or associated with theformation of neurofibrillary lesions, such as tau-associated diseases,disorders or conditions, referred to as tauopathies. According toparticular embodiments, the neurodegenerative disease, disorder, orcondition includes any of the diseases or disorders which showco-existence of tau and amyloid pathologies including, but not islimited to, Alzheimer's Disease, Parkinson's Disease, Creutzfeldt-Jacobdisease, Dementia pugilistica, Down's Syndrome,Gerstmann-Straussler-Scheinker disease, inclusion body myositis, prionprotein cerebral amyloid angiopathy, traumatic brain injury, amyotrophiclateral sclerosis, parkinsonism-dementia complex of Guam, Non-Guamanianmotor neuron disease with neurofibrillary tangles, argyrophilic graindementia, corticobasal degeneration, Dementia Lewy Amyotrophic Lateralsclerosis, diffuse neurofibrillary tangles with calcification,frontotemporal dementia, preferably frontotemporal dementia withparkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobardementia, Hallevorden-Spatz disease, multiple system atrophy,Niemann-Pick disease type C, Pick's disease, progressive subcorticalgliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,Myotonic dystrophy, chronic traumatic encephalopathy (CTE), Primaryage-related tauopathy (PART), or Lewy body dementia (LBD). According toparticular embodiments, the neurodegenerative disease, disorder, orcondition is Alzheimer's disease or another tauopathy.

The present invention also provides a method for promoting clearance oftau aggregates from the brain of a subject, said method comprisingadministering to the subject a pharmaceutical composition according toan embodiment of the invention, under conditions effective to promoteclearance of the tau aggregates from the brain of the subject. Accordingto particular embodiments, the tau aggregates are neurofibrillarytangles or their pathological tau precursors.

The present invention also provides a method for slowing progression ofa tau-pathology related behavioral phenotype in a subject, said methodcomprising administering to the subject a pharmaceutical compositionaccording to an embodiment of the invention, under conditions effectiveto slow the progression of the tau-pathology related behavioralphenotype in the subject.

In a preferred embodiment of the present invention, administration of atau peptide, via administration of a pharmaceutical compositionaccording to an embodiment of the invention, induces an active immuneresponse in the subject to the tau peptide and to the pathological formof tau, thereby facilitating the clearance of related tau aggregates,slowing the progression of tau-pathology related behavior and/ortreating the underlying tauopathy. In accordance with this aspect of thepresent invention, an immune response involves the development of abeneficial humoral (antibody mediated) response directed against the taupeptide and a cellular (mediated by antigen-specific T cells or theirsecretion products) response directed against the T-cell epitope or theimmunogenic carrier.

As used herein, a tau-pathology related behavioral phenotype includes,without limitation, cognitive impairments, early personality change anddisinhibition, apathy, abulia, mutism, apraxia, perseveration,stereotyped movements/behaviors, hyperorality, disorganization,inability to plan or organize sequential tasks, selfishness/callousness,antisocial traits, a lack of empathy, halting, agrammatic speech withfrequent paraphasic errors but relatively preserved comprehension,impaired comprehension and word-finding deficits, slowly progressivegait instability, retropulsions, freezing, frequent falls, non-levodoparesponsive axial rigidity, supranuclear gaze palsy, square wave jerks,slow vertical saccades, pseudobulbar palsy, limb apraxia, dystonia,cortical sensory loss, and tremor.

In carrying out the methods of the present invention, it is preferableto select a subject having or at risk of having Alzheimer's disease orother tauopathy, a subject having tau aggregates in the brain, or asubject exhibiting a tangle related behavioral phenotype prior toadministering the immunogenic peptides or antibodies of the presentinvention. Subjects amenable to treatment include individuals at risk ofdisease but not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease. Therefore, the presentmethods can be administered prophylactically to the general populationwithout the need for any assessment of the risk of the subject patient.The present methods are especially useful for individuals who have aknown genetic risk of Alzheimer's disease. Such individuals includethose having relatives who have experienced the disease, and those whoserisk is determined by analysis of genetic or biochemical markers.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30 years of age). Usually, however, it is not necessary to begintreatment until a patient reaches 40, 50, 60, or 70 years of age.Treatment typically entails multiple dosages over a period of time.Treatment can be monitored by assaying antibody, or activated T-cell orB-cell responses to the therapeutic agent over time. If the responsedecreases, a booster dosage is indicated.

In prophylactic applications, pharmaceutical compositions containing thetau peptides are administered to a patient susceptible to, or otherwiseat risk of, Alzheimer's disease or other tauopathy in an amountsufficient to eliminate or reduce the risk, lessen the severity, ordelay the outset of the disease, including biochemical, histologicand/or behavioral symptoms of the disease, its complications andintermediate pathological phenotypes presented during development of thedisease. In therapeutic applications, pharmaceutical compositionscontaining a tau peptide are administered to a patient suspected of, oralready suffering from, such a disease in an amount sufficient to cure,or at least partially arrest, the symptoms of the disease (biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease.

Effective doses of a pharmaceutical composition of the invention, forthe prevention and/or treatment of the neurodegenerative disease,disorder, or condition vary depending upon many different factors,including mode of administration, target site, physiological state ofthe patient, other medications administered, and whether treatment isprophylactic or therapeutic. The amount of peptides depends on whetheradjuvant is also administered, with higher dosages being required in theabsence of adjuvant. The timing of injections can vary significantlyfrom once a day, to once a year, to once a decade. A typical regimenconsists of an immunization followed by booster injections at timeintervals, such as 6 week intervals. Another regimen consists of animmunization followed by booster injections 1, 2, 6, 9 and 12 monthslater. Another regimen entails an injection every two months for life.Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

It is readily appreciated by those skilled in the art that the regimenfor the priming and boosting administrations can be adjusted based onthe measured immune responses after the administrations. For example,the boosting compositions are generally administered weeks or monthsafter administration of the priming composition, for example, about 2-3weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or26 weeks, or 28 weeks, or 30 weeks or 32 weeks or 36 weeks or one to twoyears after administration of the priming composition.

The peptides can be administered by parenteral, topical, intravenous,oral, subcutaneous, intra-arterial, intracranial, intraperitoneal,intradermal, intranasal, or intramuscular means for prophylactic and/ortherapeutic treatment. The most typical route of administration of animmunogenic agent is subcutaneous or intramuscular injection. Thislatter type of injection is most typically performed in the arm or legmuscles.

According to particular aspects, one or more boosting immunizations canbe administered. The antigens in the respective priming and boostingcompositions, however many boosting compositions are employed, need notbe identical, but should share antigenic determinants or besubstantially similar to each other.

The composition can, if desired, be presented in a kit, pack ordispenser, which can contain one or more unit dosage forms containingthe active ingredient. The kit, for example, can comprise metal orplastic foil, such as a blister pack. The kit, pack, or dispenser can beaccompanied by instructions for administration.

According to particular embodiments, the kit comprises at least one of apharmaceutical composition comprising a liposome according to anembodiment of the invention and a pharmaceutical composition comprisinga conjugate according to an embodiment of the invention.

EMBODIMENTS

The invention provides also the following non-limiting embodiments.

Embodiment 1 is a liposome, comprising:

a. a tau peptide; and

b. a helper T cell epitope;

wherein the tau peptide is presented on the surface of the liposome.

Embodiment 2 is the liposome of Embodiment 1, wherein the tau peptide isa tau phosphopeptide.

Embodiment 3 is the liposome of Embodiment 1 or 2, further comprising atoll-like receptor ligand.

Embodiment 4 is the liposome of Embodiment 3, wherein the toll-likereceptor ligand comprises at least one of a toll-like receptor 4 ligandand toll-like receptor 9 ligand.

Embodiment 5 is the liposome of Embodiment 3 or 4, wherein the toll-likereceptor ligand is a toll-like receptor 4 ligand. 100.1621 Embodiment 6is the liposome of Embodiment 5, wherein the toll-like receptor 4 ligandcomprises monophosphoryl lipid A (MPLA).

Embodiment 7 is the liposome of Embodiment 3 or 4, wherein the toll-likereceptor ligand is a toll-like receptor 9 ligand.

Embodiment 8 is the liposome of Embodiment 7, wherein the toll-likereceptor 9 ligand comprises a lipidated CpG oligonucleotide.

Embodiment 9 is the liposome of Embodiment 1, comprising:

a. a tau peptide;

b. a helper T cell epitope; and

c. at least one of

-   -   i. a toll-like receptor 9 ligand, and    -   ii. a toll-like receptor 4 ligand.

Embodiment 10 is the liposome of Embodiment 9, wherein the tau peptideis a tau phosphopeptide.

Embodiment 11 is the liposome of Embodiment 9 or 10, wherein thetoll-like receptor 9 ligand is a lipidated CpG oligonucleotide.

Embodiment 12 is the liposome of any of Embodiments 9 to 11, wherein theliposome comprises the toll-like receptor 4 ligand and toll-likereceptor 9 ligand.

Embodiment 13 is the liposome of Embodiment 12, wherein the toll-likereceptor 4 ligand comprises monophosphoryl lipid A (MPLA).

Embodiment 14 is a liposome, comprising:

a. a tau phosphopeptide;

b. a helper T-cell epitope;

c. a lipidated CpG oligonucleotide; and

d. an adjuvant containing a toll-like receptor 4 ligand;

wherein the tau phosphopeptide is presented on the surface of theliposome.

Embodiment 15 is the liposome of Embodiment 14, wherein the toll-likereceptor 4 ligand comprises monophosphoryl lipid A (MPLA).

Embodiment 16 is the liposome of any of Embodiments 1 to 15, wherein thehelper T cell epitope is encapsulated in the liposome.

Embodiment 16a is the liposome of any of Embodiments 1 to 15, whereinthe helper T cell epitope is incorporated in the membrane of theliposome.

Embodiment 16b is the liposome of any of Embodiments 1 to 15, whereinthe helper T cell epitope is presented on the surface of the liposome.

Embodiment 17 is a liposome composition, comprising:

a. a tau phosphopeptide;

b. a helper T cell epitope;

c. a lipidated CpG oligonucleotide; and

d. a monophosphoryl lipid A (MPLA);

wherein the tau phosphopeptide is presented on the surface of theliposome, and

the T-cell epitope is encapsulated in the liposome.

Embodiment 17a is the liposome of Embodiment 17, wherein the MPLA is3-O-desacyl-4′-monophosphoryl lipid A, preferably MPLTM.

Embodiment 17b is the liposome of Embodiment 17, wherein the MPLA ismonophosphoryl hexa-acyl lipid A, 3-deacyl, preferably 3D-(6-acyl)PHAD®.

Embodiment 17c is the liposome of Embodiment 17, wherein the MPLA ismonophosphoryl 3-deacyl lipid A, preferably 3D-PHAD®.

Embodiment 18 is the liposome of any of Embodiments 1 to 17c, furthercomprising one or more lipids selected from the group consisting of1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dimyristoyl-sn-glycero-3-phosphoryl-3′-rac-glycerol (DMPG), andcholesterol.

Embodiment 19 is the liposome of any of Embodiments 1 to 18, wherein thetau peptide has an amino acid sequence selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:12, or at least 85%, 90% or 95%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1 to SEQ ID NO:12.

Embodiment 19-1 is the liposome of Embodiment 19, wherein the taupeptide is a phosphopeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-3 and 5-12.

Embodiment 19-2 is the liposome of Embodiment 19-1, wherein the tauphosphopeptide comprises the amino acid sequence of SEQ ID NO:1.

Embodiment 19-3 is the liposome of Embodiment 19-1, wherein the tauphosphopeptide comprises the amino acid sequence of SEQ ID NO:2.

Embodiment 19-4 is the liposome of Embodiment 19-1, wherein the tauphosphopeptide comprises the amino acid sequence of SEQ ID NO:3.

Embodiment 19a is the liposome of any one of Embodiments 19, 19-1, 19-2,19-3 and 19-4, wherein the amino acid sequence further comprises one ormore modifications to allow the tau peptide to be presented on thesurface of the liposome.

Embodiment 19b is the liposome of Embodiment 19a, wherein the one ormore modifications comprise at least one of palmitoylation and dodecylmodification.

Embodiment 19c is the liposome of Embodiment 19a or 19b, wherein the taupeptide is modified at its N-terminus by the one or more modifications.

Embodiment 19d is the liposome of any of Embodiments 19a to 19c, whereinthe tau peptide is modified at its C-terminus by the one or moremodifications.

Embodiment 19e is the liposome of Embodiment 19d, wherein the taupeptide is palmitoylated at both of its N-terminus and C-terminus.

Embodiment 19f is the liposome of any of Embodiments 19a-19e, whereinthe tau peptide further comprises one or more additional amino acids tofacilitate the one or more modifications.

Embodiment 19g is the liposome of Embodiment 19f, wherein the one ormore additional amino acids are selected from the group consisting ofLys, Cys, Ser and Thr.

Embodiment 19h is the liposome of any of Embodiments 19 to 19g, whereinthe tau peptide is amidated at its C-terminus.

Embodiment 19i is the liposome of any of Embodiments 19 to 19h, whereinthe tau peptide consists of an amino acid sequence selected from thegroup consisting of SEQ ID NO:27 to SEQ ID NO:38.

Embodiment 19j is the liposome of any of Embodiments 19-19i, wherein thetau peptide consists of the amino acid sequence of SEQ ID NO:27.

Embodiment 19k is the liposome of any of Embodiments 19-19i, wherein thetau peptide consists of the amino acid sequence of SEQ ID NO:28.

Embodiment 19l is the liposome of any of Embodiments 19-19i, wherein thetau peptide consists of the amino acid sequence of SEQ ID NO:29.

Embodiment 20 is the liposome of any of Embodiments 1 to 19l, whereinthe helper T cell epitope comprises at least one amino acid sequenceselected from the group consisting of: SEQ ID NO:23 to SEQ ID NO:26.

Embodiment 20a is the liposome of Embodiment 20, wherein helper T cellepitope comprises at least two amino acid sequences selected from thegroup consisting of: SEQ ID NO:23 to SEQ ID NO:26.

Embodiment 20b is the liposome of Embodiment 20, wherein helper T cellepitope comprises at least three amino acid sequences selected from thegroup consisting of: SEQ ID NO:23 to SEQ ID NO:26.

Embodiment 20c is the liposome of Embodiment 20, wherein helper T cellepitope comprises the four amino acid sequences of: SEQ ID NO:23 to SEQID NO:26.

Embodiment 20d is the liposome of any of Embodiments 20a to 20c, whereinthe two or more amino acid sequences selected from the group consistingof SEQ ID NO:23 to SEQ ID NO:26 are covalently linked by a linker.

Embodiment 20e is the liposome of Embodiment 20d, wherein the linkercomprises one or more amino acids selected from the group consisting ofVal (V), Ala (A), Arg (R), Gly (G), Ser (S), Lys (K).

Embodiment 20f is the liposome of Embodiment 20e, wherein the linkercomprises an amino acid sequence selected from the group consisting ofVVR, GS, RR and RK.

Embodiment 20g is the liposome of any of Embodiments 20 to 20f, whereinthe helper T cell epitope is amidated at its C-terminus.

Embodiment 20h is the liposome of any of Embodiments 20 to 20g, whereinthe helper T cell epitope is modified for insertion into the membrane ofthe liposome, presentation on the surface of the liposome orencapsulation in the liposome, depending on the intended location of thehelper T cell epitope.

Embodiment 20i is the liposome of any of Embodiments 20 to 20h, whereinthe helper T cell epitope consisting of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:13 to SEQ ID NO:17.

Embodiment 20j is the liposome of any of Embodiments 1 to 20i, whereinliposome comprises the tau peptide and the helper T cell epitope at aweight ratio of 6:1.

Embodiment 20k is the liposome of any of Embodiments 1 to 20i, whereinliposome comprises the tau peptide and the helper T cell epitope at aweight ratio of 5:1.

Embodiment 20l is the liposome of any of Embodiments 1 to 20i, whereinliposome comprises the tau peptide and the helper T cell epitope at aweight ratio of 4:1.

Embodiment 20m is the liposome of any of Embodiments 1 to 20i, whereinliposome comprises the tau peptide and the helper T cell epitope at aweight ratio of 3:1.

Embodiment 20n is the liposome of any of Embodiments 1 to 20i, whereinliposome comprises the tau peptide and the helper T cell epitope at aweight ratio of 2:1.

Embodiment 20o is the liposome of any of Embodiments 1 to 20i, whereinliposome comprises the tau peptide and the helper T cell epitope at aweight ratio of 1:1.

Embodiment 21 is the liposome of any of Embodiments 1 to 20o, whereinthe lipidated CpG oligonucleotide comprises the nucleotide sequenceselected from the group consisting of SEQ ID NO:18 to SEQ ID NO:22.

Embodiment 21a is the liposome of Embodiment 21, wherein the CpGoligonucleotide has one or more phosphorothioate internucleotidelinkages.

Embodiment 21b is the liposome of Embodiment 21a, wherein the CpGoligonucleotide has all phosphorothioate internucleotide linkages.

Embodiment 21c is the liposome of any of Embodiments 21 to 21b, whereinlipidated CpG oligonucleotide comprises the CpG oligonucleotidecovalently linked to at least one lipophilic group via a linker.

Embodiment 21d is the liposome of Embodiment 21c, wherein the linkercomprises (C2H4O)n, wherein n is an integer of 0 to 10.

Embodiment 21e is the liposome of Embodiment 21c, wherein the linkercomprises an alkyl spacer having 3 to 12 carbons.

Embodiment 21f is the liposome of any of Embodiments 21 to 21e, whereinthe at least one lipophilic group is cholesterol.

Embodiment 21g is the liposome of any of Embodiments 21 to 21f, whereinthe lipidated CpG oligonucleotide comprises the nucleotide sequence ofSEQ ID NO:18 or SEQ ID NO:19 covalently linked to a cholesterol moleculevia a linker comprising (C2H4O)n, wherein n is an integer of 3 to 5.

Embodiment 22 is a liposome, comprising:

-   -   a. a tau peptide having an amino acid sequence selected from the        group consisting of SEQ ID NO:27 to SEQ ID NO:38;    -   b. a helper T cell epitope having an amino acid sequence        selected from the group consisting of SEQ ID NO:39 to SEQ ID        NO:44, preferably, the helper T cell epitope consisting of an        amino acid sequence selected from the group consisting of SEQ ID        NO:13 to SEQ ID NO:17;    -   c. a lipidated CpG oligonucleotide having a nucleotide sequence        selected from the group consisting of SEQ ID NO:18 to SEQ ID        NO:22, wherein the CpG oligonucleotide comprises one or more        phosphorothioate internucleotide linkages, and the CpG        oligonucleotide is covalently linked to at least one cholesterol        via a linker; and    -   d. monophosphoryl lipid A (MPLA).

Embodiment 22a is a liposome of Embodiment 22, comprising:

-   -   a. a tau phosphopeptide consisting of the amino acid sequence of        SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29;    -   b. a helper T cell epitope consisting of the amino acid sequence        of SEQ ID NO:13    -   c. a lipidated CpG oligonucleotide consisting of the nucleotide        sequence of SEQ ID NO: 18 or SEQ ID NO:19 covalently linked to a        cholesterol via a linker comprising (C2H4O)n, wherein n is an        integer of 3 to 7; and    -   d. monophosphoryl lipid A (MPLA).

Embodiment 22b is the liposome of Embodiment 22 or 22a, wherein the MPLAis 3-O-desacyl-4′-monophosphoryl lipid A, preferably MPLTM.

Embodiment 22c is the liposome of Embodiment 22 or 22a, wherein the MPLAis monophosphoryl hexa-acyl lipid A, 3-deacyl, preferably 3D-(6-acyl)PHAD®.

Embodiment 22d is the liposome of Embodiment 22 or 22a, wherein the MPLAis monophosphoryl 3-deacyl lipid A, preferably 3D-PHAD®.

Embodiment 23 is the liposome of any one of Embodiments 22 to 22d,wherein the helper T cell epitope is encapsulated in the liposome.

Embodiment 24 is a pharmaceutical composition comprising the liposome ofany of Embodiments 1 to 23 and a pharmaceutically acceptable carrier.

Embodiment 25 is a conjugate comprising a tau phosphopeptide and animmunogenic carrier conjugated thereto via a linker, having thefollowing structure:

wherein x is an integer of 0 to 10; and

n is an integer of 2 to 15.

Embodiment 25a is a conjugate comprising a tau phosphopeptide and animmunogenic carrier conjugated thereto via a linker, having thestructure of formula (II):

wherein

-   -   x is an integer of 0 to 10; and    -   n is an integer of 2 to 15.

Embodiment 26 is the conjugate of Embodiment 25 or 25a, wherein x is aninteger of 2 to 6.

Embodiment 27 is the conjugate of Embodiment 25 or 25a, wherein x is 3.

Embodiment 28 is the conjugate of any of Embodiments 25 to 25a, whereinn is 3 to 7.

Embodiment 29 is the conjugate of any of Embodiments 25 to 28, whereinthe carrier is an immunogenic carrier selected from the group consistingof keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197, and an outermembrane protein mixture from N. meningitidis (OMP), or a derivativethereof.

Embodiment 30 is the conjugate of any of Embodiments 25 to 29, whereinthe tau phosphopeptide consists of the amino acid sequence selected fromthe group consisting of SEQ ID NO:1 to SEQ ID NO:12.

Embodiment 30a is the conjugate of Embodiment 30, wherein the tauphosphopeptide consists of the amino acid sequence of SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:3.

Embodiment 31 is the conjugate of any of Embodiments 25 to 30, whereinthe carrier is CRM197.

Embodiment 32 is the conjugate of Embodiment 25, having the structureof:

wherein n is 3-7.

Embodiment 32a is the conjugate of Embodiment 25, wherein the

-   -   KLH-[m-maleimidobenzoyl-N-hydroxysuccinimide        ester—cysteine—(C₂H₄O)x-Tau peptide]_(n)

wherein

-   -   the Tau peptide consisting of SEQ ID NO:1 or SEQ ID NO:3;    -   x is an integer of 0 to 10; and    -   n is an integer of 2 to 15.

Embodiment 33 is a pharmaceutical composition comprising the conjugateof any of Embodiments 25 to 32a and a pharmaceutically acceptablecarrier.

Embodiment 33a is the pharmaceutical composition of embodiment 33,further comprising an adjuvant.

Embodiment 33b is the pharmaceutical composition of embodiment 33a,wherein the adjuvant comprises at least one of a TLR-4 ligand and aTLR-9 ligand.

Embodiment 34 is a method for inducing an immune response in a subjectsuffering from a neurodegenerative disorder, comprising administering tothe subject at least one of the pharmaceutical compositions ofEmbodiments 24 and 33 to 33b.

Embodiment 35 is the method of Embodiment 34, comprising administeringto the subject at least one of the pharmaceutical compositions ofEmbodiments 24 and 33 to 33b for priming immunization, and administeringto the subject at least one of the pharmaceutical compositions ofEmbodiments 24 and 33 to 33b for boosting immunization.

Embodiment 36 is a method for treating or preventing a neurodegenerativedisease or disorder in a subject in need thereof, comprisingadministering to the subject at least one of the pharmaceuticalcompositions of Embodiment 24 or 33.

Embodiment 37 is the method of Embodiment 36, comprising administeringto the subject at least one of the pharmaceutical compositions ofEmbodiments 24 and 33 to 33b for priming immunization, and administeringto the subject at least one of the pharmaceutical compositions ofEmbodiments 24 and 33 to 33b for boosting immunization.

Embodiment 38 is the method of any of Embodiments 34 to 37, wherein theneurodegenerative disease or disorder is caused by or associated withthe formation of neurofibrillary lesions.

Embodiment 39 is the method of any of Embodiments 34 to 38, wherein theneurodegenerative disease or disorder is Alzheimer's Disease,Parkinson's Disease, Creutzfeldt-Jacob disease, Dementia pugilistica,Down's Syndrome, Gerstmann-Sträussler-Scheinker disease, inclusion bodymyositis, prion protein cerebral amyloid angiopathy, traumatic braininjury, amyotrophic lateral sclerosis, parkinsonism-dementia complex ofGuam, Non-Guamanian motor neuron disease with neurofibrillary tangles,argyrophilic grain dementia, corticobasal degeneration, Dementia LewyAmyotrophic Lateral sclerosis, diffuse neurofibrillary tangles withcalcification, frontotemporal dementia, preferably frontotemporaldementia with parkinsonism linked to chromosome 17 (FTDP-17),frontotemporal lobar dementia, Hallevorden-Spatz disease, multiplesystem atrophy, Niemann-Pick disease type C, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,Myotonic dystrophy, chronic traumatic encephalopathy (CTE), Primaryage-related tauopathy (PART), or Lewy body dementia (LBD).

Embodiment 40 is the method of any of Embodiments 34 to 39, wherein theneurodegenerative disease or disorder is Alzheimer's disease,Parkinson's Disease, Down's Syndrome, progressive supranuclear palsy(PSP), frontotemporal dementia and parkinsonism linked to chromosome 17(FTDP-17), Pick's disease, Corticobasal Degeneration, Dementia LewyAmyotrophic Lateral sclerosis, Myotonic disphasy, chronic traumaticencephalopathy (CTE), Cerebral angiopahty, Primary age-related tauopathy(PART), or Lewy body dementia (LBD).

Embodiment 40b is the method of any of Embodiments 34 to 39, wherein theneurodegenerative disease or disorder is Alzheimer's disease,progressive supranuclear palsy (PSP), frontotemporal dementia andparkinsonism linked to chromosome 17 (FTDP-17), or Pick's disease andPART (primary age-related tauopathy).

Embodiment 40c is the method of any of Embodiments 34 to 39, wherein theneurodegenerative disease or disorder is Alzheimer's disease,Parkinson's Disease, Down's Syndrome, frontotemporal dementia andparkinsonism linked to chromosome 17 (FTDP-17), CorticobasalDegeneration, Dementia Lewy Amyotrophic Lateral sclerosis, Myotonicdisphasy, chronic traumatic encephalopathy (CTE), Cerebral angiopahty,Primary age-related tauopathy (PART), or Lewy body dementia (LBD).

Embodiment 41 is a kit comprising at least one of the pharmaceuticalcomposition of Embodiment 24 and the pharmaceutical composition ofEmbodiment 33, 33a or 33b.

Embodiment 42 is a helper T cell epitope consisting of an amino acidsequence selected from the group consisting of SEQ ID NO:13 to SEQ IDNO:17.

Embodiment 43 is a pharmaceutical composition comprising the helper Tcell epitope of Embodiment 42.

Embodiment 44 is a method of enhancing an immune response to an antigenin a subject in need thereof, comprising administering to the subjectthe antigen together with the pharmaceutical composition of Embodiment43.

EXAMPLES

The following examples of the invention are to further illustrate thenature of the invention. It should be understood that the followingexamples do not limit the invention and that the scope of the inventionis to be determined by the appended claims.

The experimental methods used in the following examples, unlessotherwise indicated, are all ordinary methods. The reagents used in thefollowing embodiments, unless otherwise indicated, are all purchasedfrom ordinary reagent suppliers.

Example 1: Preparation of Liposomal Vaccines

Preparation of the Control Liposomal Vaccine (Ethanol InjectionTechnique)

The control liposomal vaccine was produced by Ethanol (EtOH) Injectiontechnique followed by extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen,Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol(Dishman, Netherlands) and MPLA (Avanti Polar Lipids, AL, USA) weresolubilized at a molar ratio of 9:1:7:0.05 in a 20:1 (V/V) mixture ofEtOH and tert-butanol (t-BuOH) at 60° C. The lipid/ethanol solution wasdiluted in phosphate buffer saline (PBS) pH 7.4 at 60° C. to maintain10% EtOH concentration and resulting in the formation of multilamellarliposome vesicles (MLVs). The MLVs were then submitted to 5 sequentialpasses of extrusion through three polycarbonate filters (Whatman) with apore size of 0.08 um in series using Emulsiflex-05 (Avestin, Canada).The resulting liposomes were diluted in PBS pH 7.4 and heated to 60° C.to obtain a liposome solution prior to tau peptide addition.

An acetate tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2(Bachem AG, Switzerland), herein referred to as the activepharmaceutical ingredient (API), was dissolved in PBS at pH 11.4 with2.0% octyl β-D-glucopyranoside (Sigma-Aldrich, USA) at a concentrationof 1 mg/mL, and the peptide solution was injected into the liposomesolution at 60° C. followed by stirring for 30 minutes at 60° C.Concentration was done through ultrafiltration to a target final volume,and buffer exchange was carried out 10 times with PBS pH 7.4 duringdiafiltration. The resulting liposomes, with the API presented on thesurface of the liposomes, were then sterile filtered by passing throughtwo 0.2 um polycarbonate syringe filters in series, and the finalproduct was stored at 5° C.

Preparation of the Liposome X, Y, Z and Z⁺ Vaccines

The Liposome X and Y vaccines were produced by thin-lipids filmtechnology followed by homogenization and extrusion.

The Liposome Z+ vaccines, with a final API concentration of 1200 ug/mland final T50 concentration of 1200 ug/ml were produced by EthanolInjection technique followed by extrusion and the liposome Z vaccines,with a final API concentration of 400 ug/ml and final T50 concentrationof 100 ug/ml, were produced by thin-lipids film technology followed byhomogenization and extrusion.

The Liposome Z++ vaccine, with a final API concentration of 400 ug/mland final T50 concentration of 400 ug/ml, was produced by thin-lipidsfilm technology followed by homogenization and extrusion.

The Liposome Z+++ vaccine, with a final API concentration of 1200 ug/mland final T50 concentration of 300 ug/ml, were produced by EthanolInjection technique followed by extrusion.

Preparation of Liposome X, Y, Z and Z++ Vaccines by Thin Lipid FilmTechnique

The Liposome X, Y, Z and Z++ vaccines were produced by thin-lipids filmtechnology followed by homogenization and extrusion. First, DMPC (LipoidGmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany),cholesterol (Dishman, Netherlands) and monophosphoryl hexa-acyl Lipid A3-deacyl synthetic (3D-(6-acyl) PHAD®) (Avanti Polar Lipids, AL, USA)were solubilized at a molar ratio of 9:1:7:0.05 in EtOH at 60° C., withthe exception of Liposome Y, which did not contain 3D-(6-acyl) PHAD®.Ethanol was evaporated under vacuum rotavapor to obtain thin lipid film.

Lipid film was rehydrated with PBS pH 7.4, 5% DMSO (all Sigma-Aldrich)containing 0.15 mg/mL T50 peptide (Peptides & Elephants, Germany). Thesample was gently stirred for 15 min and was further vigorously vortexedto dissolve the thin lipid film. Resulting multilamellar vesicles weresubjected to 10 freeze-thaw cycles (liquid N2 and waterbath at 37° C.)and submitted to homogenization followed by sequential extrusion throughpolycarbonate membranes (Whatman, UK) with a pore size of 0.08 um. Boththe homogenization and extrusion steps were done in an EmulsiFlex-05(Avestin, Canada). Extruded liposomes with encapsulated T50 peptide wereconcentrated by ultrafiltration, and buffer was exchanged to PBS pH 7.4by diafiltration. The resulting liposomes were diluted in PBS pH 7.4 andheated to 60° C. to obtain a liposome solution prior to tau peptide andadjuvant addition.

CpG2006-Cholesterol (CpG2006-Chol) (Microsynth, Switzerland) is a DNAoligonucleotide with all internucleotide linkages as thiophosphate thatis modified at 5′ terminus with a Cholesterol molecule through aphosphate bond by means of a PEG spacer. CpG2006-Cholesterol(CpG2006-Chol) (Microsynth, Switzerland) was dissolved in PBS pH 7.4 at1 mg/mL and injected into the liposome solutions (with the exception ofLiposome X, which does not contain CpG2006-Chol) followed by incubationfor 15 minutes before insertion of the API.

The API (Bachem AG, Switzerland) was dissolved in PBS pH 11.4 with 2%Octyl B-D-glucopyranoside (Sigma-Aldrich, USA) at a concentration of 1mg/mL, and the peptide solution was injected into the liposome solutionat 60° C. followed by stirring for 30 min at 60° C. Concentration wasdone through ultrafiltration to obtain the target value (400 ug/ml APIand 100 ug/ml T50 for liposome X, Y, Z; and 400 ug/ml API and 400 ug/mlT50 for Liposome Z++), and buffer exchange was carried out 10 times withPBS pH 7.4 during diafiltration. The resulting liposomes with the APIpresented on the surface of the liposomes were then sterile filtered bypassing through 0.2 um polycarbonate syringe filters, and the finalproduct was stored at 5° C.

Preparation of Liposome O by Ethanol Injection Technique

The Liposome O vaccine was produced by Ethanol (EtOH) Injectiontechnique followed by extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen,Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol(Dishman, Netherlands) and MPLA (Avanti Polar Lipids, AL, USA) weresolubilized at a molar ratio of 9:1:7:0.05 in a 20:1 (V/V) mixture ofEtOH and tert-butanol (t-BuOH) at 60° C. The lipid/ethanol solution wasdiluted in phosphate buffer saline (PBS) pH 7.4 at 60° C. to maintain10% EtOH concentration and resulting in the formation of multilamellarliposome vesicles (MLVs). The MLVs were then submitted to 5 sequentialpasses of extrusion through three polycarbonate filters (Whatman) with apore size of 0.08 um in series using Emulsiflex-C5 (Avestin, Canada).The resulting liposomes were diluted in PBS pH 7.4 and heated to 60° C.to obtain a liposome solution prior to tau peptide addition.

T46 peptide (Pepscan, the Netherlands) was dissolved in PBS pH 7.4 at 1mg/mL and injected into the liposome solutions followed by incubationfor 15 minutes before insertion of the API.

The API (Bachem, Switzerland) was dissolved in PBS pH 11.4 with 2% OctylB-D-glucopyranoside (Sigma-Aldrich, USA) at a concentration of 1 mg/mL,and the peptide solution was injected into the liposome solution at 60°C. followed by stirring for 30 min at 60° C. Concentration was donethrough ultrafiltration to obtain the target value (400 ug/ml API and100 ug/ml T46), and buffer exchange was carried out 10 times with PBS pH7.4 during diafiltration. The resulting liposomes with the API presentedon the surface of the liposomes were then sterile filtered by passingthrough 0.2 um polycarbonate syringe filters, and the final product wasstored at 5° C.

Preparation of Liposome Z⁺ and Liposome Z⁺⁺⁺ Vaccines by EthanolInjection

The Liposome Z⁺ and Liposome Z⁺⁺⁺ vaccines were produced using anethanol injection based process. First, DMPC (Lipoid GmbH, Ludwigshafen,Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol(Dishman, Netherlands) and 3D-(6-acyl) PHAD® (Avanti Polar Lipids, AL,USA) were solubilized at a molar ratio of approximately 9:1:7:0.04 inEtOH at 60° C. T50 peptide (Bachem AG, Switzerland) was dissolved in 10mM His/270 mM sucrose (pH 5.8-6.0). Then, the lipid ethanol solution wasinjected into the solution containing T50 peptide and gently stirred for15 min, resulting in multilamellar vesicles (MLVs). MLVs were submittedto homogenization (6 times for Liposome Z⁺, and no homogenization forLiposome Z⁺⁺⁺) followed by sequential extrusion through polycarbonatemembranes (Whatman, UK) with a pore size of 0.08 um (5 passes forLiposome Z⁺, 3-5 times for Liposome Z⁺⁺⁺). Both the homogenization andextrusion steps were done in an EmulsiFlex-05 (Avestin, Canada) forLiposome Z⁺. Extrusion of Liposome Z⁺⁺⁺ was done using LIPEX filterextruder. Extruded liposomes were concentrated by ultrafiltration, andbuffer was exchanged to 20 mM His/145 mM NaCL pH 7.4 by diafiltration.The resulting liposomes with encapsulated T50 peptide were diluted in 20mM His/145 mM NaCL pH 7.4 and heated to 60° C. to obtain a liposomesolution prior to the additions of the API and the adjuvant.

CpG2006-Chol (Microsynth, Switzerland for Liposome Z+; Avecia, USA forLiposome Z+++) was dissolved in 20 mM His/145 mM NaCl pH 7.4 at 1 mg/mLand injected into the liposome solution followed by incubation for 15minutes before insertion of the API.

The API (Bachem AG, Switzerland) was dissolved in carbonate buffer pH10.2 with 1% Octyl B-D-glucopyranoside (Sigma-Aldrich, USA), at aconcentration of 1 mg/mL, and the peptide solution was injected into theliposome Z+ solution at 60° C. followed by stirring for 30 min at 60° C.The peptide solution was mixed into the liposome Z+ solution usingT-Line Mixing at 60° C. followed by stirring for 30 min at 60° C.Concentration was done through ultrafiltration to obtain the targetvalue (1200 ug/ml API and 1200 ug/ml T50 for Liposome Z+; and 1200 ug/mlAPI and 300 ug/ml T50 for Liposome Z+++), and buffer exchange wascarried out 10 times with 10 mM His/270 mM Sucrose pH 6.5 duringdiafiltration. The resulting Z+ liposomes with the API presented on thesurface of the liposomes and the resulting Z+++ liposomes with the APIpresented on the surface of the liposomes were then sterile filtered bypassing through 0.2 um polycarbonate syringe/capsule filters, and thefinal product was stored at 5° C.

Preparation of the Liposome L, M, & N Vaccines

The Liposome L, M, and N vaccines were produced by thin-lipids filmtechnology followed by homogenization and extrusion. First, DMPC (LipoidGmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany),cholesterol (Dishman, Netherlands), and MPLA (Avanti Polar Lipids, AL,USA) were solubilized at a molar ratio of 9:1:7:0.05 in EtOH at 60° C.Ethanol was evaporated under vacuum rotavapor in order to obtain thinlipid film.

Lipid film was rehydrated with PBS pH 7.4, 5% DMSO (all Sigma-Aldrich)containing either:

-   -   0.15 mg/mL T48 peptide (Peptides&Elephants, Germany)—for        Liposome M; or    -   0.13 mg/mL T50 peptide (Peptides&Elephants, Germany)—for        Liposome L; or    -   0.15 mg/mL T52 peptide (Peptides&Elephants, Germany)—for        Liposome N.

The sample was gently stirred for 15 min and was further vigorouslyvortexed to dissolve the thin lipid film. Resulting multilamellarvesicles were subjected to 10 freeze-thaw cycles (liquid N2 andwaterbath at 37° C.) and submitted to homogenization followed bysequential extrusion through polycarbonate membranes (Whatman, UK) witha pore size of 0.08 um. Both the homogenization and extrusion steps weredone in an EmulsiFlex-05 (Avestin, Canada). Extruded liposomes wereconcentrated by ultrafiltration, and buffer was exchanged to PBS pH 7.4by diafiltration. The resulting liposomes with encapsulated T48, T50 orT52 peptide were diluted in PBS pH 7.4 and heated to 60° C. to obtain aliposome solution prior to tau peptide addition.

The API (Bachem AG, Switzerland) was dissolved in PBS pH 11.4 with 2%Octyl B-D-glucopyranoside (Sigma-Aldrich, USA) at a concentration of 1mg/mL and the peptide solution was injected into the liposome solutionat 60° C. followed by stirring for 30 min at 60° C. Concentration wasdone through ultrafiltration to obtain a target value (400 ug/ml API and100 ug/ml T48, T50 or T52) and buffer exchange was carried out 10 timeswith PBS pH 7.4 during diafiltration. The resulting liposomes with theAPI presented on the surface of the liposomes were then sterile filteredby passing through 0.2 um polycarbonate syringe filters and the finalproduct was stored at 5° C.

Preparation of the Liposome R, S and T Vaccines

The Liposome R, S and T vaccines were produced using an ethanolinjection based process followed by extrusion. First, DMPC (Lipoid GmbH,Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany),cholesterol (Dishman, Netherlands) and 3D-(6-acyl) PHAD® (Avanti PolarLipids, AL, USA) were solubilized at a molar ratio of 9:1:7:0.04 in EtOHat 60° C. For Liposome R and T, the above lipid ethanol solution wasmixed to the 10 mM histidine pH 5.8 supplemented by 270 mM sucrose toreach 10% solvent (EtOH) and then incubated for 30 minutes at 60° C. ForLiposome S, T50 peptide (Bachem AG, Switzerland) was dissolved in 10 mMHis/270 mM sucrose (pH 5.8-6.0). Lipid-buffer mix related to Liposome R,S and T were gently stirred for 15 min, resulting in multilamellarvesicles (MLVs). The resulting multi lamellar vesicles were submitted toextrusion through polycarbonate membranes (Whatman, UK) with a pore sizeof 0.08 um (5×) done in an EmulsiFlex-05 high pressure system (Avestin,Canada).

Extruded liposomes were concentrated by ultrafiltration, and buffer wasexchanged to 20 mM His/145 mM NaCL pH 7.4 by diafiltration. Theresulting liposomes with encapsulated T50 for Liposome S and theresulting Liposomes R and T were further diluted in 20 mM His/145 mMNaCL pH 7.4 and heated to 60° C. to obtain a liposome solution prior tothe additions of the API and T57 for Liposome T.

For Liposome T, T57 was dissolved to 1 mg/mL in 1% OctylB-D-glucopyranoside (Sigma-Aldrich, USA) in deionized distilled waterand inserted in liposome followed by incubation for 15 minutes at 60° C.before API insertion.

The API (Bachem AG, Switzerland) was dissolved in carbonate buffer pH10.2 with 1% Octyl B-D-glucopyranoside (Sigma-Aldrich, USA), at aconcentration of 1 mg/mL, and the peptide solution was mixed into theliposome solution at 60° C. followed by stirring for 30 min at 60° C.Concentration was done through ultrafiltration to obtain the followingtarget value:

-   -   1200 ug/ml API for Liposome R;    -   1200 ug/ml API and 300 ug/ml T50 for Liposome S; and    -   1200 ug/ml API and 300 ug/ml T57 for Liposome T;

Buffer exchange was carried out 10 times with 10 mM His/270 mM SucrosepH 6.5 during diafiltration. The resulting liposomes with the APIpresented on the surface of the liposomes were then sterile filtered bypassing through 0.2 um polycarbonate syringe filters, and the finalproduct was stored at 5° C.

Example 2: Preparation of Conjugate Vaccine

Peptides and Adjuvants

Sequences of two multi-phosphorylated peptide epitopes (TAUVAC-p7.1 andTAUVAC-p22.1 which have three and two phosphorylated amino acids,respectively) were refined by optimizing the length such that they mightbetter bind surface immunoglobulin of B cells, and such that thesequences did not contain epitopes predicted to bind human HLA class IA, B, and C molecules with high affinity. The latter criterion wasimportant to avoid the induction of a cytotoxic CD8+ T cell responseagainst tau that could potentially cause significant neuronal damage.Using the T cell epitope prediction tool of the Immune Epitope Databaseand Analysis Resources, peptide TAUVAC-p7.1 showed no predicted epitopescapable of binding to human HLA class I A, B, C and HLA class II DQ andDR molecules with high affinity, while peptide TAUVAC-p22.1 waspredicted to contain epitopes binding to HLA class II DQ and DRmolecules with intermediate/high affinity (data not shown).

Phosphorylated tau peptides (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3)used in this study were produced synthetically (Pepscan, NL) with thephospho-residues added during synthesis. A conjugate comprisingphosphorylated tau peptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 3 covalently linked to a KLH carrier via a linker isherein referred to as Conjugate B or Conjugate C, respectively. Aconjugate comprising phosphorylated tau peptide having the amino acidsequence of SEQ ID NO: 2 covalently linked to a CRM carrier via a linkeris herein referred to as Conjugate A.

To manufacture Conjugates B and C, vaccine peptides were conjugated tothe carrier protein KLH via a m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS) linker and an extra cysteine on the N-terminus of thepeptide. The unbound peptide was removed using a Sephadex G25 columnbefore concentrating the conjugate. Conjugates were mixed beforeinjection to either a potent multicomponent adjuvant (Sigma AdjuvantSystem, Sigma-Aldrich) or a single component depot adjuvant (aluminumhydroxide, Alhydrogel®, Invivogen) following manufacturer'sinstructions.

Vaccine peptides were conjugated to the carrier protein CRM197 via apolyethylene glycol (PEG)-cysteine-acetamidopropionamide linker.Phosphorylated tau peptide having the amino acid sequence of SEQ ID NO:2 was produced synthetically (Polypeptide Laboratories SAS), withphospho-residues and PEG3 spacer added during synthesis. Conjugate A wasmanufactured by conjugating the carrier protein CRM197 via asuccinimidyl 3-(bromoacetamide) propionate (SBAP) linker to a cysteineon the N-terminus of the peptide. SBAP was ligated to CRM197 proteinprimary amines (—NH2) via NHS ester reaction chemistry. The excess SBAPlinker was removed using ultrafiltration and diafiltration (UF/DF). TheCRM197-SBAP intermediate was conjugated to the phosphorylated taupeptide, and once the reaction was completed, the conjugation reactionwas terminated by adding excess amount of L-cystine to quench thereaction. The crude CRM197-peptide conjugated product was purified usinga Capto Q ImpRes (GE Healthcare) chromatography column and eluted usinga salt isocratic method. The purified CRM197-peptide product was thenformulated into 20 mM Tris, 250 mM Sucrose, pH 8.1 to a concentration of0.5 mg/mL using UF/DF. The CRM197-tau peptide Drug Substance (DS) wasgenerated by adding a 10% PS80 stock buffer to reach a finalconcentration of 0.01% PS80. The solution was thoroughly mixed prior tofiltering.

Example 3: Vaccine Induced IgG Antibodies Specific to Tau Phosphopeptide

All animal experiments were approved and performed in accordance withlocal legislation on animal experiments. Rhesus macaques (Macacamulatta) were obtained from Kunming Biomed International Ltd, China,Yunnan Yinmore Bio-Tech Co. LTD, China and Yunnan Laboratory PrimatesInc., China. Animals were two to five years old at the start ofimmunization, and their minimum weight was 2.5 kg. A detailed clinicalexamination was performed prior to initiation of the treatment andweekly thereafter. Moreover, macaques were observed twice per day, andclinical signs were recorded.

Adult Rhesus macaques (n=3 males and 3 females per group) were immunizedsubcutaneously with 1800 μg of acetate tetrapalmitoylated phosphorylatedtau peptide of SEQ ID NO: 2 per dose of the control liposomal vaccine(liposome with tetrapalmitoylated phosphorylated tau peptide of SEQ IDNO: 2 and MPLA) or a liposomal vaccine according to embodiment of theapplication, e.g., Liposome Z (liposome with tetrapalmitoylatedphosphorylated tau peptide of SEQ ID NO: 2, 3D-(6-acyl) PHAD®, lipidatedCpG oligonucleotide CpG 2006 and T-cell peptide T50) or 15 μg per doseof a conjugate vaccine according to an embodiment of the invention(e.g., Conjugate A, phosphorylated tau peptide of SEQ ID NO: 2 linked toCRM197) co-injected with alum and CpG oligonucleotide CpG 2006 at days1, 29 and 85. Bleedings were performed before immunization and at days8, 22, 36, 50, 64, 78, 92, 106, 120, 134 and 148, and the sera wereisolated.

Specific IgG antibody titers were determined by ELISA, usingphosphorylated tau peptide of SEQ ID NO: 2 as the coating antigen. Serumfrom individual immunized monkey was serially diluted in assay buffer(PBS, 0.05% Tween20, 1% BSA) and applied to 96-well plates that had beencoated with the relevant peptide. After two hour incubation, sampleswere removed and plates washed in PBST (PBS, 0.05% Tween20). Antibodieswere detected using an HRP conjugated anti-monkey IgG (KPL), followed byABTS substrate (Roche). All samples were run in eight two-folddilutions, with positive and negative control samples included on eachplate. The data was expressed as geometric mean of end-point titers(last serum dilution inducing a positive response) per group.

As shown in FIG. 4, both the Liposome Z vaccine and the Conjugate Ainduced higher phosphopeptide-specific IgG titers, as compared to thecontrol liposomal vaccine.

Example 4: Vaccine Induced Antibodies Specific to Pathological TauStructures in Human Brain

All brain tissues were obtained from the Netherlands Brain Bank (NBB)and were collected from donors following signature of an informedconsent for a brain autopsy and the use of the samples as well as theirclinical information for research purposes. Paraffin sections fromnon-demented controls (healthy), Alzheimer's disease (AD), frontaltemporal dementia with tau pathology (FTD-tau), Pick's disease, primaryage-related tauopathy (PART) and progressive supranuclear palsy (PSP)were used. Brain regions included parietal cortex, middle frontal gyms,hippocampus or the caudate nucleus.

In particular, formalin-fixed paraffin embedded sections from parietalcortices of a control human subject (healthy) and a human subjectsuffering from Alzheimer's disease (AD Braak V/VI) were stained withpost-immune macaque serum diluted 1:100 in normal antibody diluent(Immunologic). The sections were then washed and stained with a Goatanti-monkey-HRP (Abcam). The staining was finally visualized using3,3′-diaminobenzidine (DAB; Dako) which deposits a brown specific stainin the presence of horse radish peroxidase (HRP). Slides werecounterstained with haematoxylin, dehydrated and mounted with Quick Dmounting medium (Klinipath). Pictures were taken with a Leica DC500microscope.

Results in FIG. 5 show that post-immune sera from Rhesus macaquesimmunized with Liposome Z (liposome with tetrapalmitoylatedphosphorylated tau peptide of SEQ ID NO: 2, 3D-(6-acyl) PHAD®, lipidatedCpG oligonucleotide CpG 2006 and T-cell peptide T50) stainedpathological tau structures in human brain sections. Serum was collectedfrom Rhesus macaques at day 106 after the primary immunization with theimproved liposomes. This macaque received immunizations at month 0, 1,and 3 prior to serum collection. The left (AD Braak V/VI) panel showsstaining of parietal cortex from a Braak Stage V donor. Arrows indicatestaining of tau tangles. The right (Healthy) panel shows staining ofparietal cortex from a Braak Stage 0 donor. Serum was applied tosections at a 1:100 dilution, followed by goat anti-monkey antibody at1:100, and staining was visualized using a DAB developer.

Results in FIG. 6 show that serum from Rhesus macaques immunized withConjugate A which contains phosphorylated tau peptide of SEQ ID NO: 2plus soluble CpG and alum hydroxide binds to pathological tau structuresin human AD brain sections. Serum was collected at day 106 after theprimary immunization with the CRM conjugate vaccine. These macaquesreceived immunizations at month 0, 1, and 3 prior to serum collection.The upper (AD) panels show staining of parietal cortex, including tautangles, from a Braak Stage V donor. The lower (CTRL) panels showstaining of parietal cortex from a Braak Stage 0 donor. Serum wasapplied to sections at a 1:100 dilution, followed by goat anti-monkeyantibody at 1:100, and staining was visualized using a DAB developer.

Example 5: Liposomal Vaccines with One or Two Adjuvants

Addition of two adjuvants in the improved liposomal vaccine increasesthe level of tau phosphopeptide-specific IgG antibody titers, as well asthe consistency of antibody response between individuals.

Adult Rhesus macaques (n=3 males and 3 females per group) were immunizedsubcutaneously at days 1, 29, 85 and 169 with 1800 μg of acetatetetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2 per doseof the control liposomal vaccine or the improved liposomal vaccine withencapsulated T50 T-cell epitope, containing either 3D-(6-acyl) PHAD®adjuvant alone (Liposome X, FIG. 7A), lipidated CpG 2006 oligonucleotideadjuvant alone (Liposome Y, FIG. 7B), or both 3D-(6-acyl) PHAD® andlipidated CpG 2006 oligonucleotide adjuvants (Liposome Z, FIG. 7C).Bleedings were performed before immunization and at days 8, 22, 36, 50,64, 78, 92, 106, 120, 134, 148, 162, 176 and 190 and the sera wereisolated. Specific IgG antibody titers in the sera were determined byELISA, using phosphorylated tau peptide of SEQ ID NO: 2 as the coatingantigen and an anti-monkey IgG secondary antibody. Resulting antibodylevels are presented as end-point titers (last serum dilution inducing apositive response) for each individual monkey over time. Eachimmunization group is represented in one panel (FIGS. 7A, 7B, and 7C).Geometric mean of end-point titers per group ±95% confidence interval ispresented in FIG. 7D. In summary, FIGS. 7A, 7B, 7C, and 7D show thatinclusion of two adjuvants in the liposomal vaccine containingencapsulated T50 improved the level and consistency of antibody responseto tau phosphopeptide, resulting in less variability in antibodyresponse among individual monkeys. More specifically, as shown in FIG.7D, improved liposomal vaccines with phosphorylated tau peptide of SEQID NO: 2, T50 T-cell epitope (Liposome X, Y and Z) and 1 or 2 adjuvants,induced higher titers against the tau phosphopeptide than the controlliposomal vaccine with no T-cell epitope. All monkeys were responderswhen injected with each of the improved liposomal vaccines, while 4 outof 6 animals were responders with the control liposomal vaccine.

Example 6: Vaccines Induced Antibodies Specific to the Enriched PairedHelical Filaments (ePHF)

Groups of Rhesus macaques (n=3 males and 3 females per group) wereimmunized subcutaneously by vaccination at day 1 and day 29 with (i) theimproved liposomal vaccine containing the T50 T-cell epitope and3D-(6-acyl) PHAD® adjuvant alone (Liposome X), (ii) the improvedliposomal vaccine containing the T50 T-cell epitope and lipidated CpG2006 adjuvant alone (Liposome Y), (iii) the improved liposomal vaccinecontaining the T50 T-cell epitope and two adjuvants (3D-(6-acyl) PHAD®and lipidated CpG2006, Liposome Z), or (iv) the conjugate vaccine(phosphorylated tau peptide of SEQ ID NO: 2 linked to CRM197)co-injected with alum and CpG oligonucleotide CpG 2006 (Conjugate A).

Preparations of enriched paired helical filaments (ePHF) were obtainedfrom post-mortem brain tissues of histologically confirmed AD subjectsby sarcosyl extraction of insoluble tau, using a modified method ofGreenberg and Davies (Greenberg and Davies, 1991, Proc Natl Acad SciUSA, 87(15):5827-31). Antibody titers specific for enriched pairedhelical filaments (ePHF) were evaluated using the Mesoscale Discovery(MSD) platform. MSD streptavidin plates were coated with thebiotinylated anti-tau capturing antibody (HT7-biotin, ThermoScientific)before incubation with ePHF isolated from Alzheimer's disease patients,while the IgG antibodies specific for ePHF were further detected using aSulfoTag-labelled anti-human IgG antibody that cross-reacts with monkeyIgG antibodies. More specifically, ePHF was added to MSD Gold small spotstreptavidin 96-well plates (MSD) previously saturated with 1% BSA andcoated with biotinylated HT-7 (Thermo Scientific). After one hour ofincubation, plates were washed with PBST and serial dilutions of serawere added and incubated for two hours. Bound antibodies were detectedusing a SulfoTag labelled anti-human IgG antibody followed by a fixationstep in 1% PFA before adding the Read Buffer T. Plates were analyzedusing a Sector Imager (MSD). Results were expressed in Arbitrary unitsper milliliter (AU/mL) for each individual monkey, together with thegeometric mean per group. Antibody titers specific for ePHF at Day 50after the first immunization are represented.

FIG. 8 shows that all of the vaccines induced high titers ofePHF-specific IgG antibodies.

Similar results with high titers of ePHF-specific IgG antibodies werealso obtained with other liposomes, such as Liposome Z+, administered toRhesus macaques via intramuscular administration.

Example 7: The Breadth of Tau Phosphopeptide-Specific Antibody Inducedby the Liposomal Vaccine and Conjugate Vaccine in Rhesus Monkeys

Groups of Rhesus macaques (n=3 males and 3 females per group) wereimmunized subcutaneously at days 1 and 29 with (i) the improvedliposomal vaccine containing encapsulated T50 and two adjuvants: TLR4ligand (3D-(6-acyl) PHAD®) and lipidated CpG 2006 oligonucleotide(Liposome Z), and (ii) the conjugate vaccine (phosphorylated tau peptideof SEQ ID NO: 2 linked to CRM) (Conjugate A) co-injected with alum andCpG oligonucleotide CpG 2006. The epitope recognition profile ofantibodies was determined by epitope mapping ELISA three weeks after thesecond immunization (Day 50) using a library of N-terminallybiotinylated 8-mer peptides, shifted by one amino acid and covering thesequence of phosphorylated tau peptide of SEQ ID NO: 2, as well as thesequence of SEQ ID NO: 4 (VYKSPVVSGDTSPRHL, non-phosphorylated taupeptide having the same amino acid sequence as SEQ ID NO: 2) and thecorresponding biotinylated full length peptides.

FIGS. 9A and 9B show that monkeys immunized with liposome Z produced IgGantibodies that bind mostly to the N-terminal part of the phosphorylatedpeptide of SEQ ID NO: 2 (FIG. 9A), whereas monkeys immunized with theconjugate vaccine (phosphorylated tau peptide of SEQ ID NO: 2 linked toCRM) generated IgG antibodies that bind mostly to the C-terminal part ofthe tau peptide of SEQ ID NO: 2 (FIG. 9B).

Example 8: Increased Titers of IgG Antibodies Specific to TauPhosphopeptide Induced by Liposomal Vaccine with Encapsulated T-CellEpitope

Three groups of C57BL/6J mice (n=10 per group) were immunizedsubcutaneously at days 0 and 14 with i) liposomal vaccine containingTLR4 agonist (3D-(6-acyl) PHAD®), (Liposome R), ii) liposomal vaccinecontaining encapsulated T-cell epitope T50 and TLR4 ligand (3D-(6-acyl)PHAD®) as adjuvant, (Liposome S), or iii) liposomal vaccine containinganchored T-cell epitope T57 on the liposomal surface (i.e.dipalmitoylated T50) and TLR4 ligand (3D-(6-acyl) PHAD®) as adjuvant,(Liposome T). Level of IgG antibodies specific to phosphorylated taupeptide of SEQ ID NO: 2 was measured 21 and 35 days after the firstinjection in mouse plasma by ELISA; results were presented as values ofindividual mice, together with the geometric mean per group ±95% CIexpressed in arbitrary units (AU) per mL. As shown in FIG. 10A,vaccination with the liposomal vaccine containing encapsulated T50(Liposome S) induced significantly higher antibody titers than thecontrol liposomal vaccine (Liposome R) and the liposomal vaccinecontaining anchored T-cell epitope (Liposome T) 21 days after the firstimmunization (Kruskal-Wallis test: p=0.0089 and p=0002, respectively)and also higher antibody titers than the control liposomal vaccine andsignificantly higher antibody titers than the liposomal vaccinecontaining anchored T-cell epitope 35 days after the first immunization(Kruskal-Wallis test: p=0.7591 and p=0053, respectively) (FIG. 10B).

Example 9: Liposomal Vaccines Induced T-Cell Response Specific to theIncorporated T-Cell Epitope

Three groups of C57BL/6J mice (n=5 per group) were immunizedsubcutaneously at days 0, 14 and 28 with (i) the improved liposomalvaccine with encapsulated T-cell peptide T48 (containing T-cell epitopesPADRE, T2, T30 and T17 separated with the GS linker,) and a TLR4 agonistas adjuvant (MPLA) (Liposome M), (ii) the improved liposomal vaccinewith encapsulated T52 (containing T-cell epitopes PADRE, T2 and T30separated with the RK linker) and a TLR4 agonist (MPLA) as adjuvant(Liposome N) or (iii) PBS. Spleens from mice were harvested 42 daysafter the first immunization for the analysis of T-cell responses byIL-4 and IFN-γ ELISPOT. Single cell suspensions were incubated withmedium, T48 or T52 peptide at 10 ug/mL for 48 hours. Plates wereincubated with a biotinylated anti-mouse IL-4 or IFN-γ monoclonalantibodies and with streptavidin alkaline phosphatase (AP). Spots weredeveloped by adding the AP substrate. FIGS. 11A and 11B show that therestimulation of mouse splenocytes with the same peptide as the oneencapsulated in the liposome induced IL-4 (FIG. 11B) and IFN-y spotforming cells (FIG. 11A), while the splenocytes of PBS-injected mice didnot. This confirmed that the addition of T-cell epitope to the vaccineinduced the activation of specific T-cells, allowing them to furtherprovide the help in antibody production to the tau-specific B-cells.

Example 10: Liposomal Vaccines Containing Encapsulated T-Cell Epitopeand Anchored T-Cell Epitope

Groups of Rhesus macaques (n=6 per group) were immunized subcutaneouslyat days 1, 29, 85 and 169 with (i) liposomal vaccine containingencapsulated T-cell epitope T50 and TLR4 ligand (MPLA) as adjuvant(liposome L), (ii) liposomal vaccine containing anchored T-cell epitopeT46 and TLR4 ligand (MPLA) as adjuvant (liposome 0) and (iii) controlliposomal vaccine containing a TLR4 ligand (MPLA) as adjuvant and noT-cell epitope. Bleedings were performed before immunization (at day−14) and at days 8, 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162, 176and 190, and the sera were isolated. Specific IgG antibody titers weredetermined by ELISA, using phosphorylated tau peptide of SEQ ID NO: 2 asthe coating antigen and an anti-monkey IgG secondary antibody. Resultingantibody levels were calculated as end-point titers (last serum dilutioninducing a positive response), and the data was expressed as geometricmean per group. As shown in FIG. 12, liposomal vaccine containing anencapsulated T-cell epitope (Liposome L) and liposomal vaccinecontaining an anchored T-cell epitope (Liposome 0) each induced highertau phosphopeptide-specific antibody titers than the control liposomalvaccine without T-cell epitope.

Example 11: Antibody Response in Mice Induced by Conjugate Vaccine

Female BALB/c mice (14 mice per group) were immunized with Conjugate Bor Conjugate C (containing SEQ ID NO:1 or SEQ ID NO: 3 covalently linkedto KLH) following the schedule depicted in FIG. 13A and using thevaccine candidates adjuvated with either a potent multicomponentadjuvant (Sigma Adjuvant System®, Sigma-Aldrich, from here on referredto as Ribi) or a single component depot adjuvant (Alhydrogel® adjuvant2% or aluminum hydroxide gel, InvivoGen, from here on referred to asalum). The amino acid sequence of SEQ ID NO:1 contains only one aminoacid difference compared to that in the mouse protein, while thesequence of SEQ ID NO:3 is 100% conserved between humans and mice. Thus,the selected epitopes can be reasonably considered “self” proteins formice and mice should be a relevant model to investigate the limitationsthat immune tolerance might place on immunogenicity.

As a first measure of vaccine immunogenicity flow cytometry was used tomeasure induction of T follicular helper cells (TfHs) in the cervicallymph nodes draining the vaccine injection site (four mice per group).TfHs are a specialized population of CD4+ T cells characterized by theexpression of CXCR5, PD-1 and ICOS among other molecules. TfHs expandafter exposure to a vaccine or other immune stimuli and support affinitymaturation of B cells in the germinal center (Crotty, 2011, AnnualReviews of Immunology. Vol 29:p621-663). The number of TfHs inducedcorrelates positively with the protective efficacy of vaccines in humans(Bentebibel et al., 2013, Sci Transl Med., 5(176):176ra32; Spensieri etal., 2013, Proc Natl Acad Sci USA., 110(35):14330-5) and small animals.As shown in FIG. 13B, both vaccines, as well as the KLH plus adjuvantcontrol immunization induced measurable TfHs in vaccinated mice.Moreover, all the animals receiving active vaccine (Conjugate B andConjugate C groups) or active placebo (KLH) plus alum, had significantlymore TfHs than the animals given an inactive placebo (PBS group), whendraining cervical lymph nodes were harvested seven days after the firstimmunization (P=0.0044 for KLH-TAUVAC-p7.1; P=0.0482 forKLH-TAUVAC-p22.1; P=0.0063 for KLH, using an ANOVA test followed byDunnett's adjustment for multiple comparisons).

ELISA was conducted to determine the serum titer of antibodies bindingto the tau phosphopeptides and to KLH at Day 0 and at four additionaltime points after immunization (days 14, 28, 56, and 84, see FIGS. 13C,13D, 13G and 13H). As shown in FIG. 13C, immunization with Conjugate Binduced binding antibodies reactive against the corresponding vaccinepeptide. For animals immunized with Conjugate B and Ribi adjuvant,binding titers against the vaccine peptide were significantly higherthan the binding titers induced by the active placebo (compare ConjugateB plus Ribi to KLH plus Ribi) at all time points measured (P<0.001 usingan ANOVA test followed by Tukey's adjustment for multiple comparisons).For the alum adjuvanted group the difference was significant only atdays 56 and 84 (P=0.001 and 0.012 respectively).

The tau specific antibody response to Conjugate C (FIG. 13D) was ofoverall lower magnitude than was the response to Conjugate B, althoughthe assay differences (different coating peptide) preclude making adirect statistical comparison between the two vaccines. Nonetheless,antibody titers against Conjugate C were significantly higher in micevaccinated with Conjugate C plus Ribi than in mice receiving activeplacebo KLH Ribi at days 28 and 84 (P=0.001 and 0.008 respectively)after immunization; titers of the alum adjuvanted group were notsignificantly different than those of the active placebo.

Although the carrier protein protects the phosphopeptide fromdegradation in vivo to some extent, it was likely that phosphatasedigestion of the peptide antigens in vivo would expose somenon-phosphorylated peptide to the immune system. To determine whetherthat exposure resulted in generation of antibodies capable of bindingnon-phosphorylated peptide in Conjugate B and Conjugate C, ELISA wasperformed using non-phosphorylated peptides as the coating antigen. Asshown in FIGS. 13E and 13F, the response to non-phosphorylated taupeptides was low, comparable to the response of the active placebos tothe same non-phosphorylated peptide. Moreover, in animals immunized withConjugate B and Ribi, binding titers against the phosphorylated peptidewere significantly higher than the binding titers against thenon-phosphorylated peptide at all timepoints measured (P=0.009 at day14; P<0.0001 at day 28, 56 and 84 using an ANOVA test). For the alumadjuvanted group the difference was significant only at days 56 and 84(P=0.0002 and 0.001 respectively). For animals immunized with ConjugateC, responses to the phosphorylated peptide were higher only when Ribiadjuvant was used (P<0.0001 at day 28; P=0.0001 at day 56 and 84).

Example 12: Antibodies Induced by Conjugate Vaccines Bind toPhysiologically Relevant Forms of Altered Tau

To further determine whether the vaccine induced antibodies could bindto physiologically relevant forms of altered tau, we used post-immunesera from vaccinated mice to stain post-mortem human brain sectionscollected either from Alzheimer's disease patients (5 AD cases), frompatients affected by other tauopathies (3 cases of PART, FTD, PICK andPSP), or from age-matched healthy controls (5 control cases, CTRL). Asexpected, sera from control animals (PBS and active placebo groups) didnot bind the brain sections, while ATB, a monoclonal antibody that bindsto pTau [pSer202, pThr 205] obtained from a murine clone, showed strongimmunoreactivity of tau pathology in an adjacent tissue section of thecorresponding area (FIG. 14). Sera from animals immunized with theactive vaccines Conjugate B and Conjugate C bound pathological taustructures not only in the AD sections (data not shown), but also inthose from other tauopathies (FIG. 14). Conjugate B induced antibodiesreacted with (pre-)tangles, neuropil threads and neuritic plaques in ADcases. These post-immune sera were also able to immunoreact withneurofibril tangles and neuropil threads in PART brain tissue, neuronalinclusions and neuropil threads in FTD-tau (MAPT P301S) tissue,inclusions and astrocytes in some of the Pick's disease cases andfinally the neuronal inclusions, neuropil threads and astrocytes typicalof PSP. Conjugate C induced polyclonal sera also reacted to pathologicaltau structures characteristics of each tauopathy. In the AD cases, thestaining was mainly focused on neurofibrillary tangles, and to lessextent on neuritic plaques and neuropil threads. Lower magnification ofthe corresponding areas showed similar results (Data not known).

Example 13: Vaccine-Induced Antibodies are Functional in Mice

The protective efficacy of Conjugate B vaccine was tested in aninjection model of tauopathy (Peeraer et al., 2015, Neurobiol Dis.,73:83-95). In this model, mice made susceptible to tauopathy via agenetic mutation (P301L) receive an intracerebral injection of enrichedPHF isolated from human AD brain following the timelines indicated inFIG. 15A. The injection, which is performed before the onset oftransgene-induced tauopathy, accelerates the development of tauopathy inthese animals. Conversely, when the ePHF “seed” is pre-mixed with anantibody capable of suppressing tau seeding activity like ATB, theinduction of tauopathy is reduced (unpublished data, not shown).

Following the scheme in FIG. 15A, we assessed the development oftauopathy after stereotaxic injection of enriched human PHF pre-mixedwith IgG purified from serum of animals immunized with Conjugate B, Ribior with the active control KLH Ribi. Two months after the injection, thebrains of these mice were harvested and the amount of aggregated tau intotal and sarkosyl-insoluble fractions was determined using standardbiochemical analysis. Data obtained showed that when mice were injectedwith ePHF that had been pre-mixed with IgG from mice vaccinated withConjugate B, there was significantly less aggregated phospho-tau in bothtotal (FIG. 15B) and sarkosyl-insoluble (FIG. 15C) fractions compared toanimals receiving the control injection (p<0.0001 KLH Ribi vsKLH-TAUVAC-p7.1 Ribi using an ANOVA test followed by Holm-Bonferroniadjustment for multiple comparisons). The sarkosyl-insoluble tau beingwell accepted to correlate with the pathological features of tauopathy,this result demonstrates that antibodies induced by vaccination withKLH-TAUVAC-p7.1 are protective in vivo.

Example 14: Vaccine-Induced Antibodies are Functional in Non-HumanPrimates

Rhesus macaques were immunized with alum and CpG oligonucleotideadjuvated Conjugate B (n=6) or with KLH (n=2) at day 1, 29, 85 and 169.Blood was collected every 14 days and sera from animals immunized withConjugate B tested for reactivity on the immunizing peptide using ELISA(FIG. 16A) and human ePHF using MSD (FIG. 16B). Immunization withConjugate B resulted in a sustained and consistent antibody responseagainst the vaccine phosphopeptide. Moreover, all animals had measurableantibody levels against human ePHF with 3 out of 6 animals showing highreactivity on this antigen. Sera collected from animals 50 daysfollowing primary immunization were applied to human brain sections fromhealthy individuals or from AD patients (FIG. 16C). Post-immune serafrom Conjugate B group stained pathological tau structures, namelyneurofibrillary tangles, neuropil threads and neuritic plaques in ADbrain tissue, while sera from KLH-immunized mice did not show anyreactivity. No staining was observed on control tissue. When tested inthe tau immunodepletion assay, animals receiving Conjugate B hadantibodies able to bind and deplete tau seed (p=0.03 at day 50 using anANOVA test followed by Dunnett's adjustment for multiple comparisons),while immunization with KLH did not trigger such antibodies (FIG. 16D).Pre- and post-immunization sera were also tested in the neutralizationassay as serially diluted individual samples (FIG. 16E). Changes frombaseline (CFB) were calculated as difference between FRET counts forreadings at day −14 prior to vaccination (baseline) and post vaccinationdays 50, 106 and 190 respectively. Response at a specific postvaccination day (day,) was then computed as follows:

Response=% FRET_day_(i)−% FRET_baseline

A general linear mixed model on aforementioned responses, with animal asrandom effect, was applied with variables vaccine groups, day and serumlevels treated as categorical variables and all their interactions.Given the exploratory nature of the study, no multiple testingadjustment was considered. Hypothesis testing was performed at the 5%level of significance.

Example 15: Mice Immunized with the Conjugate Vaccine in Combination ofAlum and CpG Oligonucleotide Adjuvants Resulted in Higher Titer AntibodyResponses to the Vaccine Peptide

Adult female C57BL/6 mice (n=5-6 per group) were immunizedintramuscularly with either 2 ug (FIG. 17A) or 0.2 ug (FIG. 17B) of theConjugate A vaccine. The conjugate vaccine was either administered alone(no adjuvant), with alum hydroxide, with CpG oligonucleotide, or withalum and CpG oligonucleotide combined. All mice received a primaryimmunization on day 0 of the study followed by a single boosterimmunization on day 28. The dose for the alum adjuvant was 500 ug permouse per injection, and the dose for the CpG oligonucleotide adjuvantwas 20 ug/mouse per injection. The graphs in FIGS. 17A and 17B show theresults of binding ELISA using serum collected from mice beforeimmunization (day 0) and at two time points after immunization (day 28and 42) with vaccine peptide T3.5 as the coating antigen. T3.5 specificmean endpoint titers per group were plotted, with error barsrepresenting standard error. The tables show the statistical analysis ofthe results, in which antibody titers were compared using thenon-parametric Kruskal-Wallis Test, and pairwise group comparisons wereassessed using the Wilcoxon Signed Rank test as post-hoc to the KruskalWallis test.

The results shown in FIGS. 17A and 17B illustrate that at both doses,the non-adjuvanted vaccine failed to induce a strong immune response.Use of alum or CpG oligonucleotide or a combination of both improved themagnitude of the antibody response (p<0.0152). Moreover, for animalsimmunized with 2 ug of vaccine, the adjuvant combination gavesignificantly higher antibody titers than single adjuvants at day 28(p=0.0028). The combination alum-CpG oligonucleotide also performedbetter than CpG oligonucleotide alone for animals immunized with 0.2 ugof vaccine at day 42 (p=0.0497). These data support the use of the alumand CpG oligonucleotide adjuvant combination.

Example 16: Liposomal Vaccines with Different Ratios Tau Peptide: T-CellEpitopes Induce High and Sustained Level of Tau Phosphopeptide-SpecificIgG Antibody Titers

Adult Rhesus macaques (n=6 per group) were immunized subcutaneously atdays 1, 29, 85 and 169 with 1800 μg of acetate tetrapalmitoylatedphosphorylated tau peptide of SEQ ID NO: 2 per dose in the improvedliposomal vaccine with encapsulated T50 T-cell epitope, containing both3D-(6-acyl) PHAD® and lipidated CpG 2006 oligonucleotide adjuvant with:i) 400 ug/mL of phosphorylated tau peptide of SEQ ID NO: 2 and 100 ug/mLof T50 (Liposome Z), ii) 1200 ug/mL of phosphorylated tau peptide of SEQID NO: 2 and 1200 ug/mL of T50 (Liposome Z⁺), iii) 400 ug/mL ofphosphorylated tau peptide of SEQ ID NO: 2 and 400 ug/mL of T50(Liposome Z⁺⁺), iv) 1200 ug/mL of phosphorylated tau peptide of SEQ IDNO: 2 and 300 ug/mL of T50 (Liposome Z⁺⁺⁺). Bleedings were performedbefore immunization and at days 8, 22, 36, 50, 64, 78, 92, 106, 120,134, 148, 162, 176 and 190 and the sera were isolated. Specific IgGantibody titers in the sera were determined by ELISA, usingphosphorylated tau peptide of SEQ ID NO: 2 as the coating antigen and ananti-monkey IgG secondary antibody. Resulting antibody levels werecalculated as end-point titers (last serum dilution inducing a positiveresponse) for each individual monkey over time. Geometric mean ofend-point titers per group ±95% confidence interval is presented in FIG.18, showing that all four tested liposomal vaccines induced high andsustained titers against the tau phospho-peptide.

SEQUENCE LISTING phospho-tau peptide (7.1) SEQ ID NO: 1GDRSGYS[pS]PG[pS]PG[pT]PGSRSRT SEQ ID NO: 2-phospho-tau peptide (T3.5)VYK[pS]PVVSGDT[pS]PRHL SEQ ID NO: 3-phospho-tau peptide (22.1)SSTGSIDMVD[pS]PQLA[pT]LA SEQ ID NO: 4-tau peptide VYKSPVVSGDTSPRHLSEQ ID NO: 5-phospho-tau peptide RENAKAKTDHGAEIVYK[pS]PVVSGDT[pS]PRHLSEQ ID NO: 6-phospho-tau peptide RQEFEVMEDHAGT[pY]GLSEQ ID NO: 7-phospho-tau peptide PGSRSR[pT]P[pS]LPTPPTRSEQ ID NO: 8-phospho-tau peptide GYSSPG[pS]PG[pT]PGSRSRSEQ ID NO: 9-phospho-tau peptide GDT[pS]PRHL[pS]NVSSTGSIDSEQ ID NO: 10-phospho-tau peptide PG[pS]PG[pT]PGSRSR[pT]P[pS]LPSEQ ID NO: 11-phospho-tau peptide HL[pS]NVSSTGSIDSEQ ID NO: 12-phospho-tau peptide VSGDT[pS]PRHLSEQ ID NO: 13-T50 T cell epitopeAKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLE-NH₂SEQ ID NO: 14-T46 T cell epitopeAKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEK(Pal)K (Pal)-NH₂SEQ ID NO: 15-T48 helper T cell epitopeAKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEGSLINSTKIYSYFPSVISKVNQ-NH₂ SEQ ID NO: 16-T51 helper T cell epitopeAKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSFWLRVPKVSASHLE-NH₂SEQ ID NO: 17-T52 helper T cell epitopeAKFVAAWTLKAAARKQYIKANSKFIGITELRKFNNFTVSFWLRVPKVSASHLE-NH₂SEQ ID NO: 18-CpG 2006 (also known as CpG 7909)5′-tcgtcgttttgtcgttttgtcgtt-3′wherein lower case means phosphorothioate (ps) intemucleotide linkagesSEQ ID NO: 19- CpG 1018 5′-tgactgtgaacgttcgagatga-3′wherein lower case means phosphorothioate internucleotide linkagesSEQ ID NO: 20-CpG2395 5′-tcgtcgttttcggcgcgcgccg-3′wherein lower case means phosphorothioate internucleotide linkagesSEQ ID NO: 21-CpG2216 5′-ggGGGACGATCGTCgggggg-3′wherein lower case means phosphorothioate internucleotide linkages and capital lettersmeans phosphodiester (po) linkages SEQ ID NO: 22-CpG23365′-gggGACGACGTCGTGgggggg-3′,wherein lower case means phosphorothioate internucleotide linkages and capital lettersmeans phosphodiester linkagesSEQ ID NO: 23-Pan DR epitope (PADRE) peptide AKFVAAWTLKAAASEQ ID NO: 24-P2 QYIKANSKFIGITEL SEQ ID NO: 25-P30 ENNFTVSEWLRVPKVSASHLESEQ ID NO: 26-TT₅₈₆₋₆₀₅ LINSTKIYSYFPSVISKVNQSEQ ID NO: 27-palmitoylated phospho-tau peptide (palmitoylated 7.1)K(pal)K(pal)GDRSGYS[pS]PG[pS]PG[pT]PGSRSRTK(pal)K(pal)SEQ ID NO: 28-palmitoylated phospho-tau peptide (T3, palmitoylated T3.5)K(pal)K(pal)VYK[pS]PVVSGDT[pS]PRHLK(pal)K(pal)SEQ ID NO: 29-palmitoylated phospho-tau peptide (palmitoylated 22.1)K(pal)K(pal)SSTGSIDMVD[pS]PQLA[pT]LAK(pal)K(pal)SEQ ID NO: 30-palmitoylated tau peptideK(pal)K(pal)VYKSPVVSGDTSPRHLK(pal)K(pal)SEQ ID NO: 31-palmitoylated phospho-tau peptideK(pal)K(pal)RENAKAKTDHGAEIVYK[pS]PVVSGDT[pS]PRHLK(pal)K(pal)SEQ ID NO: 32-palmitoylated phospho-tau peptideK(pal)K(pal)RQEFEVMEDHAGT[pY]GLK(pal)K(pal)SEQ ID NO: 33-palmitoylated phospho-tau peptideK(pal)K(pal)PGSRSR[pT]P[pS]LPTPPTRK(pal)K(pal)SEQ ID NO: 34-palmitoylated phospho-tau peptideK(pal)K(pal)GYSSPG[pS]PG[pT]PGSRSRK(pal)K(pal)SEQ ID NO: 35-palmitoylated phospho-tau peptideK(pal)K(pal)GDT[pS]PRHL[pS]NVSSTGSIDK(pal)K(pal)SEQ ID NO: 36-palmitoylated phospho-tau peptideK(pal)K(pal)PG[pS]PG[pT]PGSRSR[pT]P[pS]LPK(pal)K(pal)SEQ ID NO: 37-palmitoylated phospho-tau peptideK(pal)K(pal)HL[pS]NVSSTGSIDK(pal)K(pal)SEQ ID NO: 38-palmitoylated phospho-tau peptideK(pal)K(pal)VSGDT[pS]PRHLK(pal)K(pal)SEQ ID NO: 39-T50 without the C-terminal amideAKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLESEQ ID NO: 40-T46 without the -Lys(Pal)-Lys(Pal)-NH₂ at the C-terminalAKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLESEQ ID NO: 41-T48 without the C-terminal amideAKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEGSLINSTKIYSYFPSVISKVNQ SEQ ID NO: 42-T51 without the C-terminal amideAKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSFWLRVPKVSASHLESEQ ID NO: 43-T52 without the C-terminal amideAKFVAAWTLKAAARKQYIKANSKFIGITELRKFNNFTVSFWLRVPKVSASHLE SEQ ID NO: 44- T57AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLE-K(Pal)K(Pal)-NH2

REFERENCES

Asuni A A et al, J Neurosci. 2007 Aug. 22; 27(34):9115-29

-   Bentebibel et al., 2013, Sci Transl Med., 5(176):176ra32-   Crotty, 2011, Annual Reviews of Immunology. Vol 29:p621-663-   Friedhoff et al., Biochimica et Biophysica Acta 1502 (2000) 122-132-   Greenberg and Davies, 1991, Proc Natl Acad Sci USA, 87(15):5827-31-   Hanger et al., Trends Mol Med. 15:112-9, 2009-   Hickman et al., J. Biol. Chem. vol. 286, NO. 16, pp. 13966-13976,    Apr. 22, 2011-   Kontsekova E et al., Alzheimers Res Ther. 2014 Aug. 1; 6(4):44-   Novak P et al., Lancet Neurology 2017, 16:123-134-   Peeraer et al., 2015, Neurobiol Dis., 73:83-95-   Ries et al., 2015, Org. Biomol. Chem., 13:9673-   Spensieri et al., 2013, Proc Natl Acad Sci USA., 110(35):14330-5-   Theunis C et al., PLoS One. 2013; 8(8): e72301-   U.S. Pat. No. 7,741,297-   U.S. Pat. No. 8,647,631-   U.S. Pat. No. 9,687,447-   WO90/14837-   WO2010/115843

We claim:
 1. A conjugate comprising a tau phosphopeptide and animmunogenic carrier conjugated thereto via a linker, the conjugatehaving the structure of:

wherein x is an integer of 0 to 10; and n is an integer of 3 to
 15. 2.The conjugate of claim 1, wherein the immunogenic carrier is selectedfrom the group consisting of keyhole limpet hemocyanin (KLH), tetanustoxoid, CRM197 and an outer membrane protein mixture from N.meningitidis (OMP), or a derivative thereof.
 3. The conjugate of claim1, wherein the tau peptide has an amino acid sequence selected from thegroup consisting of SEQ ID NO:1 to SEQ ID NO:12.
 4. The conjugate ofclaim 1, wherein x is 2 to
 6. 5. The conjugate of claim 1, wherein n is3 to
 12. 6. The conjugate of claim 4, wherein x is
 3. 7. The conjugateof claim 4, wherein n is 3 to
 12. 8. The conjugate of claim 6, wherein nis 3 to
 12. 9. The conjugate of claim 7, wherein the tau peptideconsists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQID NO:3.
 10. The conjugate of claim 9, wherein the conjugate has thestructure of formula (I) and the immunogenic carrier is CRM197.
 11. Theconjugate of claim 9, wherein the conjugate has the structure of formula(II) and the immunogenic carrier is KLH.
 12. A pharmaceuticalcomposition comprising the conjugate of claim 1 and a pharmaceuticallyacceptable carrier.
 13. The pharmaceutical composition of claim 12,further comprising an adjuvant.
 14. The pharmaceutical composition ofclaim 13, wherein the adjuvant comprises at least one of an aluminumsalt and a CpG.
 15. The pharmaceutical composition of claim 14, whereinthe adjuvant comprises the aluminum salt and the CpG.
 16. Apharmaceutical composition comprising the conjugate of claim 10 and apharmaceutically acceptable carrier.
 17. A pharmaceutical compositioncomprising the conjugate of claim 11 and a pharmaceutically acceptablecarrier.
 18. A conjugate having the structure of:

wherein n is an integer of 3 to
 7. 19. A pharmaceutical compositioncomprising the conjugate of claim 18 and a pharmaceutically acceptablecarrier.
 20. The pharmaceutical composition of claim 19, furthercomprising an adjuvant.
 21. The pharmaceutical composition of claim 20,wherein the adjuvant comprises at least one of an aluminum salt and aCpG.
 22. The pharmaceutical composition of claim 21, wherein theadjuvant comprises the aluminum salt and the CpG.
 23. A conjugate havingthe structure of:

wherein the Tau peptide consisting of the amino acid sequence of SEQ IDNO:1 or SEQ ID NO:3; x is an integer of 0 to 10; and n is an integer of2 to
 15. 24. A pharmaceutical composition comprising the conjugate ofclaim 23 and a pharmaceutically acceptable carrier.
 25. Thepharmaceutical composition of claim 24, further comprising an adjuvant.26. The pharmaceutical composition of claim 25, wherein the adjuvantcomprises at least one of an aluminum salt and a CpG.
 27. Thepharmaceutical composition of claim 26, wherein the adjuvant comprisesthe aluminum salt and the CpG.
 28. A method for inducing an immuneresponse in a subject suffering from a neurodegenerative disorder,comprising administering to the subject the pharmaceutical compositionof claim
 12. 29. A method for treating or preventing a neurodegenerativedisease or disorder in a subject in need thereof, comprisingadministering to the subject the pharmaceutical composition of claim 12,wherein the neurodegenerative disease or disorder is caused by orassociated with the formation of neurofibrillary lesions.